Anti-pd-1 antibodies and uses thereof

ABSTRACT

This disclosure relates to anti-PD-1 (Programmed Cell Death Protein 1) antibodies, antigen-binding fragments, and the uses thereof.

CLAIM OF PRIORITY

This application is a divisional of U.S. application Ser. No.16/919,846, filed on Jul. 2, 2020, which claims priority toInternational Application No. PCT/CN2018/077016, filed on Feb. 23, 2018.The entire contents of the foregoing are incorporated herein byreference.

TECHNICAL FIELD

This disclosure relates to anti-PD-1 (Programmed Cell Death Protein 1)antibodies and uses thereof.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext form in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 45124-0016002_SL.txt. The text file is 34.0 KB,and was created and submitted electronically via EFS-Web on May 24,2021.

BACKGROUND

Cancer is currently one of the diseases that have the highest humanmortality. According to the World Health Organization statistical data,in 2012, the number of global cancer incidence and death cases reached14 million and 8.2 million, respectively. In China, the newly diagnosedcancer cases are 3.07 million, and the death toll is 2.2 million.

Recent clinical and commercial success of anticancer antibodies hascreated great interest in antibody-based therapeutics. There is a needto develop anti-cancer antibodies for use in various antibody-basedtherapeutics to treat cancers.

SUMMARY

This disclosure relates to anti-PD-1 antibodies, antigen-bindingfragment thereof, and the uses thereof.

In one aspect, the disclosure relates to an antibody or antigen-bindingfragment thereof that binds to PD-1 (Programmed Cell Death Protein 1)comprising: a heavy chain variable region (VH) comprisingcomplementarity determining regions (CDRs) 1, 2, and 3, wherein the VHCDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 regioncomprises an amino acid sequence that is at least 80% identical to aselected VH CDR2 amino acid sequence, and the VH CDR3 region comprisesan amino acid sequence that is at least 80% identical to a selected VHCDR3 amino acid sequence; and a light chain variable region (VL)comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises anamino acid sequence that is at least 80% identical to a selected VL CDR1amino acid sequence, the VL CDR2 region comprises an amino acid sequencethat is at least 80% identical to a selected VL CDR2 amino acidsequence, and the VL CDR3 region comprises an amino acid sequence thatis at least 80% identical to a selected VL CDR3 amino acid sequence,wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and theselected VL CDRs, 1, 2, and 3 amino acid sequences are one of thefollowing:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12,respectively;

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 7, 13, 14, respectively, and the selected VL CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 10, 11, 15,respectively.

In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acidsequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VLcomprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ IDNOs: 4, 5, and 6, respectively.

In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acidsequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VLcomprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ IDNOs: 10, 11, and 12, respectively.

In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acidsequences set forth in SEQ ID NOs: 7, 13, and 14, respectively, and theVL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQID NOs: 10, 11, and 15, respectively.

In some embodiments, the antibody or antigen-binding fragmentspecifically binds to human PD-1. In some embodiments, the antibody orantigen-binding fragment is a humanized antibody or antigen-bindingfragment thereof. In some embodiments, the antibody or antigen-bindingfragment is a single-chain variable fragment (scFV).

In another aspect, the disclosure relates to a nucleic acid comprising apolynucleotide encoding a polypeptide comprising:

-   -   (1) an immunoglobulin heavy chain or a fragment thereof        comprising a heavy chain variable region (VH) comprising        complementarity determining regions (CDRs) 1, 2, and 3        comprising the amino acid sequences set forth in SEQ ID NOs: 1,        2, and 3, respectively, and wherein the VH, when paired with a        light chain variable region (VL) comprising the amino acid        sequence set forth in SEQ ID NO: 29, 30, 31, or 40, binds to        PD-1;    -   (2) an immunoglobulin light chain or a fragment thereof        comprising a VL comprising CDRs 1, 2, and 3 comprising the amino        acid sequences set forth in SEQ ID NOs: 4, 5, and 6,        respectively, and wherein the VL, when paired with a VH        comprising the amino acid sequence set forth in SEQ ID NO: 26,        27, 28, or 39, binds to PD-1;    -   (3) an immunoglobulin heavy chain or a fragment thereof        comprising a heavy chain variable region (VH) comprising CDRs 1,        2, and 3 comprising the amino acid sequences set forth in SEQ ID        NOs: 7, 8, and 9, respectively, and wherein the VH, when paired        with a light chain variable region (VL) comprising the amino        acid sequence set forth in SEQ ID NO: 36, 37, 38 or 42, binds to        PD-1;    -   (4) an immunoglobulin light chain or a fragment thereof        comprising a VL comprising CDRs 1, 2, and 3 comprising the amino        acid sequences set forth in SEQ ID NOs: 10, 11, and 12,        respectively, and wherein the VL, when paired with a VH        comprising the amino acid sequence set forth in SEQ ID NO: 32,        33, 34, 35 or 41, binds to PD-1;    -   (5) an immunoglobulin heavy chain or a fragment thereof        comprising a heavy chain variable region (VH) comprising CDRs 1,        2, and 3 comprising the amino acid sequences set forth in SEQ ID        NOs: 7, 13, and 14, respectively, and wherein the VH, when        paired with a light chain variable region (VL) comprising the        amino acid sequence set forth in SEQ ID NO: 44 binds to PD-1;    -   (6) an immunoglobulin light chain or a fragment thereof        comprising a VL comprising CDRs 1, 2, and 3 comprising the amino        acid sequences set forth in SEQ ID NOs: 10, 11, and 15,        respectively, and wherein the VL, when paired with a VH        comprising the amino acid sequence set forth in SEQ ID NO: 43        binds to PD-1.

In some embodiments, the nucleic acid comprises a polynucleotideencoding a polypeptide comprising an immunoglobulin heavy chain or afragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprisingthe amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3,respectively.

In some embodiments, the nucleic acid comprises a polynucleotideencoding a polypeptide comprising an immunoglobulin light chain or afragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprisingthe amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6,respectively.

In some embodiments, the nucleic acid comprises a polynucleotideencoding a polypeptide comprising an immunoglobulin heavy chain or afragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprisingthe amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9,respectively.

In some embodiments, the nucleic acid comprises a polynucleotideencoding a polypeptide comprising an immunoglobulin light chain or afragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprisingthe amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12,respectively.

In some embodiments, the nucleic acid comprises a polynucleotideencoding a polypeptide comprising an immunoglobulin heavy chain or afragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprisingthe amino acid sequences set forth in SEQ ID NOs: 7, 13, and 14,respectively.

In some embodiments, the nucleic acid comprises a polynucleotideencoding a polypeptide comprising an immunoglobulin light chain or afragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprisingthe amino acid sequences set forth in SEQ ID NOs: 10, 11, and 15,respectively.

In some embodiments, the VH when paired with a VL specifically binds tohuman PD-1, or the VL when paired with a VH specifically binds to humanPD-1.

In some embodiments, the immunoglobulin heavy chain or the fragmentthereof is a humanized immunoglobulin heavy chain or a fragment thereof,and the immunoglobulin light chain or the fragment thereof is ahumanized immunoglobulin light chain or a fragment thereof.

In some embodiments, the nucleic acid encodes a single-chain variablefragment (scFv). In some embodiments, the nucleic acid is cDNA.

In one aspect, the disclosure relates to a vector comprising one or moreof the nucleic acids as described herein. In some embodiments, thevector encodes the VL region and the VH region that together bind toPD-1.

In one aspect, the disclosure provides a pair of vectors, wherein eachvector comprises one of the nucleic acids as described herein, whereintogether the pair of vectors encodes the VL region and the VH regionthat together bind to PD-1.

In another aspect, the disclosure relates to a cell comprising thevector as described herein, or the pair of vectors as described herein.In some embodiments, the cell is a CHO cell.

In one aspect, the disclosure also provides a cell comprising one ormore of the nucleic acids as described herein.

In another aspect, the disclosure provides a cell comprising two of thenucleic acids as described herein. In some embodiments, the two nucleicacids together encode the VL region and the VH region that together bindto PD-1.

In another aspect, the disclosure relates to methods of producing anantibody or an antigen-binding fragment thereof. The methods include thesteps of

-   -   (a) culturing the cell as described herein under conditions        sufficient for the cell to produce the antibody or the        antigen-binding fragment; and    -   (b) collecting the antibody or the antigen-binding fragment        produced by the cell.

In one aspect, the disclosure relates to an antibody or antigen-bindingfragment thereof that binds to PD-1 comprising a heavy chain variableregion (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region(VL) comprising an amino acid sequence that is at least 90% identical toa selected VL sequence, wherein the selected VH sequence and theselected VL sequence are one of the following:

-   -   (1) the selected VH sequence is SEQ ID NOs: 26, 27, 28, or 39,        and the selected VL sequence is SEQ ID NOs: 29, 30, 31, or 40;    -   (2) the selected VH sequence is SEQ ID NOs: 32, 33, 34, 35 or        41, and the selected VL sequence is SEQ ID NOs: 36, 37, 38 or        42;    -   (3) the selected VH sequence is SEQ ID NO: 43, and the selected        VL sequence is SEQ ID NO: 44.    -   In some embodiments, the VH comprises the sequence of SEQ ID NO:        26 and the VL comprises the sequence of SEQ ID NO: 31.

In some embodiments, the VH comprises the sequence of SEQ ID NO: 27 andthe VL comprises the sequence of SEQ ID NO: 31.

In some embodiments, the antibody or antigen-binding fragmentspecifically binds to human PD-1.

In some embodiments, the antibody or antigen-binding fragment is ahumanized antibody or antigen-binding fragment thereof. In someembodiments, the antibody or antigen-binding fragment is a single-chainvariable fragment (scFV).

In one aspect, the disclosure relates to an antibody-drug conjugatecomprising the antibody or antigen-binding fragment thereof as describedherein covalently bound to a therapeutic agent. In some embodiments, thetherapeutic agent is a cytotoxic or cytostatic agent.

In another aspect, the disclosure relates to methods of treating asubject having cancer. The methods include the steps of administering atherapeutically effective amount of a composition comprising theantibody or antigen-binding fragment thereof as described herein, or theantibody-drug conjugate as described herein, to the subject.

In some embodiments, the subject has a solid tumor. In some embodiments,the cancer is unresectable melanoma or metastatic melanoma. In someembodiments, the cancer is non-small cell lung cancer (NSCLC), squamouscell carcinoma of the head and neck (SCCHN), head and neck cancer, renalcell carcinoma (RCC), melanoma, bladder cancer, gastric cancer,urothelial cancer, Merkel-cell carcinoma, triple-negative breast cancer(TNBC), or colorectal carcinoma.

In one aspect, the disclosure relates to methods of decreasing the rateof tumor growth. The methods include the steps of contacting a tumorcell with an effective amount of a composition comprising an antibody orantigen-binding fragment thereof as described herein, or theantibody-drug conjugate as described herein.

In another aspect, the disclosure relates methods of killing a tumorcell. The methods include the steps of contacting a tumor cell with aneffective amount of a composition comprising the antibody orantigen-binding fragment thereof as described herein, or theantibody-drug conjugate as described herein.

In one aspect, the disclosure provides a pharmaceutical compositioncomprising the antibody or antigen-binding fragment thereof as describedherein, and a pharmaceutically acceptable carrier.

In another aspect, the disclosure also provides a pharmaceuticalcomposition comprising the antibody drug conjugate as described herein,and a pharmaceutically acceptable carrier.

As used herein, the term “cancer” refers to cells having the capacityfor autonomous growth. Examples of such cells include cells having anabnormal state or condition characterized by rapidly proliferating cellgrowth. The term is meant to include cancerous growths, e.g., tumors;oncogenic processes, metastatic tissues, and malignantly transformedcells, tissues, or organs, irrespective of histopathologic type or stageof invasiveness. Also included are malignancies of the various organsystems, such as respiratory, cardiovascular, renal, reproductive,hematological, neurological, hepatic, gastrointestinal, and endocrinesystems; as well as adenocarcinomas which include malignancies such asmost colon cancers, renal-cell carcinoma, prostate cancer and/ortesticular tumors, non-small cell carcinoma of the lung, and cancer ofthe small intestine. Cancer that is “naturally arising” includes anycancer that is not experimentally induced by implantation of cancercells into a subject, and includes, for example, spontaneously arisingcancer, cancer caused by exposure of a patient to a carcinogen(s),cancer resulting from insertion of a transgenic oncogene or knockout ofa tumor suppressor gene, and cancer caused by infections, e.g., viralinfections. The term “carcinoma” is art recognized and refers tomalignancies of epithelial or endocrine tissues. The term also includescarcinosarcomas, which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures. The term “sarcoma” is art recognizedand refers to malignant tumors of mesenchymal derivation. The term“hematopoietic neoplastic disorders” includes diseases involvinghyperplastic/neoplastic cells of hematopoietic origin. A hematopoieticneoplastic disorder can arise from myeloid, lymphoid or erythroidlineages, or precursor cells thereof.

As used herein, the term “antibody” refers to any antigen-bindingmolecule that contains at least one (e.g., one, two, three, four, five,or six) complementary determining region (CDR) (e.g., any of the threeCDRs from an immunoglobulin light chain or any of the three CDRs from animmunoglobulin heavy chain) and is capable of specifically binding to anepitope. Non-limiting examples of antibodies include: monoclonalantibodies, polyclonal antibodies, multi-specific antibodies (e.g.,bi-specific antibodies), single-chain antibodies, chimeric antibodies,human antibodies, and humanized antibodies. In some embodiments, anantibody can contain an Fc region of a human antibody. The term antibodyalso includes derivatives, e.g., bi-specific antibodies, single-chainantibodies, diabodies, linear antibodies, and multi-specific antibodiesformed from antibody fragments.

As used herein, the term “antigen-binding fragment” refers to a portionof a full-length antibody, wherein the portion of the antibody iscapable of specifically binding to an antigen. In some embodiments, theantigen-binding fragment contains at least one variable domain (e.g., avariable domain of a heavy chain or a variable domain of light chain).Non-limiting examples of antibody fragments include, e.g., Fab, Fab′,F(ab′)₂, and Fv fragments.

As used herein, the term “human antibody” refers to an antibody that isencoded by an endogenous nucleic acid (e.g., rearranged humanimmunoglobulin heavy or light chain locus) present in a human. In someembodiments, a human antibody is collected from a human or produced in ahuman cell culture (e.g., human hybridoma cells). In some embodiments, ahuman antibody is produced in a non-human cell (e.g., a mouse or hamstercell line). In some embodiments, a human antibody is produced in abacterial or yeast cell. In some embodiments, a human antibody isproduced in a transgenic non-human animal (e.g., a bovine) containing anunrearranged or rearranged human immunoglobulin locus (e.g., heavy orlight chain human immunoglobulin locus).

As used herein, the term “chimeric antibody” refers to an antibody thatcontains a sequence present in at least two different antibodies (e.g.,antibodies from two different mammalian species such as a human and amouse antibody). A non-limiting example of a chimeric antibody is anantibody containing the variable domain sequences (e.g., all or part ofa light chain and/or heavy chain variable domain sequence) of anon-human (e.g., mouse) antibody and the constant domains of a humanantibody. Additional examples of chimeric antibodies are describedherein and are known in the art.

As used herein, the term “humanized antibody” refers to a non-humanantibody which contains minimal sequence derived from a non-human (e.g.,mouse) immunoglobulin and contains sequences derived from a humanimmunoglobulin. In non-limiting examples, humanized antibodies are humanantibodies (recipient antibody) in which hypervariable (e.g., CDR)region residues of the recipient antibody are replaced by hypervariable(e.g., CDR) region residues from a non-human antibody (e.g., a donorantibody), e.g., a mouse, rat, or rabbit antibody, having the desiredspecificity, affinity, and capacity. In some embodiments, the Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human (e.g., mouse) immunoglobulin residues. In someembodiments, humanized antibodies may contain residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications can be made to further refine antibody performance. Insome embodiments, the humanized antibody contains substantially all ofat least one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops (CDRs) correspond to thoseof a non-human (e.g., mouse) immunoglobulin and all or substantially allof the framework regions are those of a human immunoglobulin. Thehumanized antibody can also contain at least a portion of animmunoglobulin constant region (Fc), typically, that of a humanimmunoglobulin. Humanized antibodies can be produced using molecularbiology methods known in the art. Non-limiting examples of methods forgenerating humanized antibodies are described herein.

As used herein, the term “single-chain antibody” refers to a singlepolypeptide that contains at least two immunoglobulin variable domains(e.g., a variable domain of a mammalian immunoglobulin heavy chain orlight chain) that is capable of specifically binding to an antigen.Non-limiting examples of single-chain antibodies are described herein.

As used herein, the term “multimeric antibody” refers to an antibodythat contains four or more (e.g., six, eight, or ten) immunoglobulinvariable domains. In some embodiments, the multimeric antibody is ableto crosslink one target molecule (e.g., PD-1) to at least one secondtarget molecule (e.g., CTLA-4) on the surface of a mammalian cell (e.g.,a human T-cell).

As used herein, the terms “subject” and “patient” are usedinterchangeably throughout the specification and describe an animal,human or non-human, to whom treatment according to the methods of thepresent invention is provided. Veterinary and non-veterinaryapplications are contemplated by the present invention. Human patientscan be adult humans or juvenile humans (e.g., humans below the age of 18years old). In addition to humans, patients include but are not limitedto mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, andprimates. Included are, for example, non-human primates (e.g., monkey,chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils,hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniaturepig), equine, canine, feline, bovine, and other domestic, farm, and zooanimals.

As used herein, when referring to an antibody, the phrases “specificallybinding” and “specifically binds” mean that the antibody interacts withits target molecule (e.g., PD-1) preferably to other molecules, becausethe interaction is dependent upon the presence of a particular structure(i.e., the antigenic determinant or epitope) on the target molecule; inother words, the reagent is recognizing and binding to molecules thatinclude a specific structure rather than to all molecules in general. Anantibody that specifically binds to the target molecule may be referredto as a target-specific antibody. For example, an antibody thatspecifically binds to a PD-1 molecule may be referred to as aPD-1-specific antibody or an anti-PD-1 antibody.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to polymers of amino acids of any lengthof at least two amino acids.

As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and“nucleic acid sequence” are used interchangeably herein to refer topolymers of nucleotides of any length of at least two nucleotides, andinclude, without limitation, DNA, RNA, DNA/RNA hybrids, andmodifications thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a flow chart showing the first part of an exemplary protocolof making anti-hPD-1 antibodies.

FIG. 2 is a flow chart showing the second part of an exemplary protocolof making anti-hPD-1 antibodies.

FIG. 3 is a set of flow cytometry graphs showing that the anti-hPD-1antibodies block the binding between hPD-1 and hPD-L1.

FIG. 4 is a set of graphs showing flow cytometry results of analyzingthe anti-hPD-1 antibodies' cross-reactivity with monkey PD-1 (rmPD-1),mouse PD-1 (mPD-1), and human-mouse chimeric PD-1 (chiPD-1). NC standsfor negative control.

FIG. 5 is a graph showing the results of surface plasma resonance (SPR)using the chimeric anti-hPD-1 antibody 1A7-mHvKv-IgG4-S228P and humanPD-1.

FIG. 6 is a graph showing the results of surface plasma resonance (SPR)using the chimeric anti-hPD-1 antibody 3F1-mHvKv-IgG4-S228P and humanPD-1.

FIG. 7 is a graph showing body weight over time of humanized PD-1 mice(B-hPD-1) with MC-38 tumor cells treated with mouse anti-hPD-1antibodies and Keytruda. PS stands for physiological saline.

FIG. 8 is a graph showing percentage change of body weight over time ofhumanized PD-1 mice (B-hPD-1) with MC-38 tumor cells treated with mouseanti-hPD-1 antibodies and Keytruda. PS stands for physiological saline.

FIG. 9 is a graph showing tumor size over time in humanized PD-1 mice(B-hPD-1) with MC-38 tumor cells treated with mouse anti-hPD-1antibodies and Keytruda. PS stands for physiological saline.

FIG. 10 is a graph showing body weight over time of humanized PD-1 mice(B-hPD-1) with MC-38 tumor cells treated with chimeric anti-hPD-1antibodies and Keytruda. PS stands for physiological saline.

FIG. 11 is a graph showing percentage change of body weight over time ofhumanized PD-1 mice (B-hPD-1) with MC-38 tumor cells treated withchimeric anti-hPD-1 antibodies and Keytruda. PS stands for physiologicalsaline.

FIG. 12 is a graph showing tumor size over time in humanized PD-1 mice(B-hPD-1) with MC-38 tumor cells treated with chimeric anti-hPD-1antibodies and Keytruda. PS stands for physiological saline.

FIG. 13 is a graph showing body weight over time of humanized PD-1 mice(B-hPD-1) with MC-38 tumor cells treated with humanized anti-hPD-1antibodies and Keytruda. PS stands for physiological saline.

FIG. 14 is a graph showing percentage change of body weight over time ofhumanized PD-1 mice (B-hPD-1) with MC-38 tumor cells treated withhumanized anti-hPD-1 antibodies and Keytruda. PS stands forphysiological saline.

FIG. 15 is a graph showing tumor size over time in humanized PD-1 mice(B-hPD-1) with MC-38 tumor cells treated with humanized anti-hPD-1antibodies and Keytruda. PS stands for physiological saline.

FIG. 16 lists CDR sequences of mouse anti-hPD-1 antibodies (25-1A7,18-3F1, 3-6G1) and CDR sequences of humanized anti-hPD-1 antibodiesthereof as defined by Kabat numbering.

FIG. 17 lists CDR sequences of mouse anti-hPD-1 antibodies (25-1A7,18-3F1, 3-6G1) and CDR sequences of humanized anti-hPD-1 antibodiesthereof as defined by Chothia numbering.

FIG. 18 lists amino acid sequences of human PD-1 (hPD-1), mouse PD-1(mPD-1), monkey PD-1 (rmPD-1), and chimeric PD-1 (chiPD-1).

FIG. 19 lists amino acid sequences of heavy chain variable regions andlight chain variable regions of humanized anti-hPD-1 antibodies.

FIG. 20 lists the amino acid sequence of the heavy chain variableregions and light chain variable regions of mouse anti-hPD-1 antibodies25-1A7, 18-3F1, and 3-6G1.

DETAILED DESCRIPTION

The present disclosure provides examples of antibodies, antigen-bindingfragment thereof, that bind to PD-1 (Programmed Cell Death Protein 1;also known as CD279).

PD-1 and Cancer

The immune system can differentiate between normal cells in the body andthose it sees as “foreign”, which allows the immune system to attack theforeign cells while leaving the normal cells alone. This mechanismsometimes involves proteins called immune checkpoints. Immunecheckpoints are molecules in the immune system that either turn up asignal (co-stimulatory molecules) or turn down a signal.

Checkpoint inhibitors can prevent the immune system from attackingnormal tissue and thereby preventing autoimmune diseases. Many tumorcells also express checkpoint inhibitors. These tumor cells escapeimmune surveillance by co-opting certain immune-checkpoint pathways,particularly in T cells that are specific for tumor antigens (Creelan,Benjamin C. “Update on immune checkpoint inhibitors in lung cancer.”Cancer Control 21.1 (2014): 80-89). Because many immune checkpoints areinitiated by ligand-receptor interactions, they can be readily blockedby antibodies against the ligands and/or their receptors.

PD-1 (programmed death-1) is an immune checkpoint and guards againstautoimmunity through a dual mechanism of promoting apoptosis (programmedcell death) in antigen-specific T-cells in lymph nodes whilesimultaneously reducing apoptosis in regulatory T cells(anti-inflammatory, suppressive T cells).

PD-1 is mainly expressed on the surfaces of T cells and primary B cells;two ligands of PD-1 (PD-L1 and PD-L2) are widely expressed inantigen-presenting cells (APCs). The interaction of PD-1 with itsligands plays an important role in the negative regulation of the immuneresponse. Inhibition the binding between PD-1 and its ligand can makethe tumor cells exposed to the killing effect of the immune system, andthus can reach the effect of killing tumor tissues and treating cancers.

PD-L1 is expressed on the neoplastic cells of many different cancers. Bybinding to PD-1 on T-cells leading to its inhibition, PD-L1 expressionis a major mechanism by which tumor cells can evade immune attack. PD-L1over-expression may conceptually be due to 2 mechanisms, intrinsic andadaptive. Intrinsic expression of PD-L1 on cancer cells is related tocellular/genetic aberrations in these neoplastic cells. Activation ofcellular signaling including the AKT and STAT pathways results inincreased PD-L1 expression. In primary mediastinal B-cell lymphomas,gene fusion of the MHC class II transactivator (CIITA) with PD-L1 orPD-L2 occurs, resulting in over expression of these proteins.Amplification of chromosome 9p23-24, where PD-L1 and PD-L2 are located,leads to increased expression of both proteins in classical Hodgkinlymphoma. Adaptive mechanisms are related to induction of PD-L1expression in the tumor microenvironment. PD-L1 can be induced onneoplastic cells in response to interferon γ. In microsatelliteinstability colon cancer, PD-L1 is mainly expressed on myeloid cells inthe tumors, which then suppress cytotoxic T-cell function.

The use of PD-1 blockade to enhance anti-tumor immunity originated fromobservations in chronic infection models, where preventing PD-1interactions reversed T-cell exhaustion. Similarly, blockade of PD-1prevents T-cell PD-1/tumor cell PD-L1 or T-cell PD-1/tumor cell PD-L2interaction, leading to restoration of T-cell mediated anti-tumorimmunity.

A detailed description of PD-1, and the use of anti-PD-1 antibodies totreat cancers are described, e.g., in Topalian, Suzanne L., et al.“Safety, activity, and immune correlates of anti-PD-1 antibody incancer.” New England Journal of Medicine 366.26 (2012): 2443-2454;Hirano, Fumiya, et al. “Blockade of B7-H1 and PD-1 by monoclonalantibodies potentiates cancer therapeutic immunity.” Cancer research65.3 (2005): 1089-1096; Raedler, Lisa A. “Keytruda (pembrolizumab):first PD-1 inhibitor approved for previously treated unresectable ormetastatic melanoma.” American health & drug benefits 8.Spec Feature(2015): 96; Kwok, Gerry, et al. “Pembrolizumab (Keytruda).” (2016):2777-2789; US 20170247454; U.S. Pat. Nos. 9,834,606 B; and 8,728,474;each of which is incorporated by reference in its entirety.

The present disclosure provides several anti-PD-1 antibodies,antigen-binding fragments thereof, and methods of using these anti-PD-1antibodies and antigen-binding fragments to inhibit tumor growth and totreat cancers.

Antibodies and Antigen Binding Fragments

The present disclosure provides anti-PD-1 antibodies and antigen-bindingfragments thereof. In general, antibodies (also called immunoglobulins)are made up of two classes of polypeptide chains, light chains and heavychains. A non-limiting antibody of the present disclosure can be anintact, four immunoglobulin chain antibody comprising two heavy chainsand two light chains. The heavy chain of the antibody can be of anyisotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype includingIgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chaincan be a kappa light chain or a lambda light chain. An antibody cancomprise two identical copies of a light chain and two identical copiesof a heavy chain. The heavy chains, which each contain one variabledomain (or variable region, V_(H)) and multiple constant domains (orconstant regions), bind to one another via disulfide bonding withintheir constant domains to form the “stem” of the antibody. The lightchains, which each contain one variable domain (or variable region,V_(L)) and one constant domain (or constant region), each bind to oneheavy chain via disulfide binding. The variable region of each lightchain is aligned with the variable region of the heavy chain to which itis bound. The variable regions of both the light chains and heavy chainscontain three hypervariable regions sandwiched between more conservedframework regions (FR).

These hypervariable regions, known as the complementary determiningregions (CDRs), form loops that comprise the principle antigen bindingsurface of the antibody. The four framework regions largely adopt abeta-sheet conformation and the CDRs form loops connecting, and in somecases forming part of, the beta-sheet structure. The CDRs in each chainare held in close proximity by the framework regions and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingregion.

Methods for identifying the CDR regions of an antibody by analyzing theamino acid sequence of the antibody are well known, and a number ofdefinitions of the CDRs are commonly used. The Kabat definition is basedon sequence variability, and the Chothia definition is based on thelocation of the structural loop regions. These methods and definitionsare described in, e.g., Martin, “Protein sequence and structure analysisof antibody variable domains,” Antibody engineering, Springer BerlinHeidelberg, 2001. 422-439; Abhinandan, et al. “Analysis and improvementsto Kabat and structurally correct numbering of antibody variabledomains,” Molecular immunology 45.14 (2008): 3832-3839; Wu, T. T. andKabat, E. A. (1970) J. Exp. Med. 132: 211-250; Martin et al., MethodsEnzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16(October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January. 1998);Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko andBourne, BMC Structural Biology 7:64 (2007); each of which isincorporated herein by reference in its entirety. Unless specificallyindicated in the present disclosure, Kabat numbering is used in thepresent disclosure as a default.

The CDRs are important for recognizing an epitope of an antigen. As usedherein, an “epitope” is the smallest portion of a target moleculecapable of being specifically bound by the antigen binding domain of anantibody. The minimal size of an epitope may be about three, four, five,six, or seven amino acids, but these amino acids need not be in aconsecutive linear sequence of the antigen's primary structure, as theepitope may depend on an antigen's three-dimensional configuration basedon the antigen's secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule(e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses(IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in theirconstant region, particularly in their hinges and upper CH2 domains. Thesequences and differences of the IgG subclasses are known in the art,and are described, e.g., in Vidarsson, et al, “IgG subclasses andallotypes: from structure to effector functions.” Frontiers inimmunology 5 (2014); Irani, et al. “Molecular properties of human IgGsubclasses and their implications for designing therapeutic monoclonalantibodies against infectious diseases.” Molecular immunology 67.2(2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecularanalysis of structure, function and regulation. Elsevier, 2016; each ofwhich is incorporated herein by reference in its entirety.

The antibody can also be an immunoglobulin molecule that is derived fromany species (e.g., human, rodent, mouse, camelid). Antibodies disclosedherein also include, but are not limited to, polyclonal, monoclonal,monospecific, polyspecific antibodies, and chimeric antibodies thatinclude an immunoglobulin binding domain fused to another polypeptide.The term “antigen binding domain” or “antigen binding fragment” is aportion of an antibody that retains specific binding activity of theintact antibody, i.e., any portion of an antibody that is capable ofspecific binding to an epitope on the intact antibody's target molecule.It includes, e.g., Fab, Fab′, F(ab′)₂, and variants of these fragments.Thus, in some embodiments, an antibody or an antigen binding fragmentthereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody,a bispecific scFv, a diabody, a linear antibody, a single-chain antibodymolecule, a multi-specific antibody formed from antibody fragments, andany polypeptide that includes a binding domain which is, or ishomologous to, an antibody binding domain. Non-limiting examples ofantigen binding domains include, e.g., the heavy chain and/or lightchain CDRs of an intact antibody, the heavy and/or light chain variableregions of an intact antibody, full length heavy or light chains of anintact antibody, or an individual CDR from either the heavy chain or thelight chain of an intact antibody.

In some embodiments, the antigen binding fragment can form a part of achimeric antigen receptor (CAR). In some embodiments, the chimericantigen receptor are fusions of single-chain variable fragments (scFv)as described herein, fused to CD3-zeta transmembrane- and endodomain. Insome embodiments, the chimeric antigen receptor also comprisesintracellular signaling domains from various costimulatory proteinreceptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimericantigen receptor comprises multiple signaling domains, e.g.,CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in oneaspect, the disclosure further provides cells (e.g., T cells) thatexpress the chimeric antigen receptors as described herein.

In some embodiments, the scFV has one heavy chain variable domain, andone light chain variable domain.

Anti-PD-1 Antibodies and Antigen-Binding Fragments

The disclosure provides antibodies and antigen-binding fragments thereofthat specifically bind to PD-1. The antibodies and antigen-bindingfragments described herein are capable of binding to PD-1 and canpromote PD-1 signaling pathway thus increase immune response. Thedisclosure provides e.g., mouse anti-PD-1 antibodies 25-1A7 (“1A7”),18-3F1 (“3F1”), and 3-6G1 (“6G1”), the chimeric antibodies thereof, andthe humanized antibodies thereof (e.g., antibodies as shown in Table 2).

The CDR sequences for 1A7, and 1A7 derived antibodies (e.g., humanizedantibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs:1-3, and CDRs of the light chain variable domain, SEQ ID NOs: 4-6 asdefined by Kabat numbering. The CDRs can also be defined by Chothiasystem. Under the Chothia numbering, the CDR sequences of the heavychain variable domain are set forth in SEQ ID NOs: 16, 17, 3 and CDRsequences of the light chain variable domain are set forth in SEQ IDNOs: 4-6.

Similarly, the CDR sequences for 3F1, and 3F1 derived antibodies includeCDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs ofthe light chain variable domain, SEQ ID NOs: 10-12, as defined by Kabatnumbering. Under Chothia numbering, the CDR sequences of the heavy chainvariable domain are set forth in SEQ ID NOs: 18, 19, 9, and CDRs of thelight chain variable domain are set forth in SEQ ID NOs: 10-12.

The CDR sequences for 6G1, and 6G1 derived antibodies include CDRs ofthe heavy chain variable domain, SEQ ID NOs: 7, 13, 14, and CDRs of thelight chain variable domain, SEQ ID NOs: 10, 11, 15, as defined by Kabatnumbering. Under Chothia numbering, the CDR sequences of the heavy chainvariable domain are set forth in SEQ ID NOs: 20, 21, 14, and CDRs of thelight chain variable domain are set forth in SEQ ID NOs: 10, 11, 15.

The amino acid sequences for heavy chain variable regions and lightvariable regions of the humanized antibodies are also provided. As thereare different ways to humanize a mouse antibody (e.g., a sequence can bemodified with different amino acid substitutions), the heavy chain andthe light chain of an antibody can have more than one version ofhumanized sequences. The amino acid sequences for the heavy chainvariable regions of humanized 1A7 antibody are set forth in SEQ ID NOs:26-28. The amino acid sequences for the light chain variable regions ofhumanized 1A7 antibody are set forth in SEQ ID NOs: 29-31. Any of theseheavy chain variable region sequences (SEQ ID NO: 26-28) can be pairedwith any of these light chain variable region sequences (SEQ ID NO:29-31).

Similarly, the amino acid sequences for the heavy chain variable regionof humanized 3F1 antibody are set forth in SEQ ID NOs: 32-35. The aminoacid sequences for the light chain variable region of humanized 3F1antibody are set forth in SEQ ID NOs: 36-38. Any of these heavy chainvariable region sequences (SEQ ID NO: 32-35) can be paired with any ofthese light chain variable region sequences (SEQ ID NO: 36-38).

As shown in FIG. 19, humanization percentage means the percentageidentity of the heavy chain or light chain variable region sequence ascompared to human antibody sequences in International ImmunogeneticsInformation System (IMGT) database. The top hit means that the heavychain or light chain variable region sequence is closer to a particularspecies than to other species. For example, top hit to human means thatthe sequence is closer to human than to other species. Top hit to humanand Macaca fascicularis means that the sequence has the same percentageidentity to the human sequence and the Macaca fascicularis sequence, andthese percentages identities are highest as compared to the sequences ofother species. In some embodiments, humanization percentage is greaterthan 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, or 95%. A detailed description regarding how to determinehumanization percentage and how to determine top hits is known in theart, and is described, e.g., in Jones, Tim D., et al. “The INNs and outsof antibody nonproprietary names.” MAbs. Vol. 8. No. 1. Taylor &Francis, 2016, which is incorporated herein by reference in itsentirety. A high humanization percentage often has various advantages,e.g., more safe and more effective in humans, more likely to betolerated by a human subject, and/or less likely to have side effects.

Furthermore, in some embodiments, the antibodies or antigen-bindingfragments thereof described herein can also contain one, two, or threeheavy chain variable region CDRs selected from the group of SEQ ID NOs:1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 7, 13, 14, SEQ ID NOs: 16, 17, 3, SEQID NOs: 18, 19, 9, and SEQ ID NOs: 20, 21, 14; and/or one, two, or threelight chain variable region CDRs selected from the group of SEQ ID NOs:4-6, SEQ ID NOs 10-12, and SEQ ID NOs: 10, 11, 15.

In some embodiments, the antibodies can have a heavy chain variableregion (VH) comprising complementarity determining regions (CDRs) 1, 2,3, wherein the CDR1 region comprises or consists of an amino acidsequence that is at least 80%, 85%, 90%, or 95% identical to a selectedVH CDR1 amino acid sequence, the CDR2 region comprises or consists of anamino acid sequence that is at least 80%, 85%, 90%, or 95% identical toa selected VH CDR2 amino acid sequence, and the CDR3 region comprises orconsists of an amino acid sequence that is at least 80%, 85%, 90%, or95% identical to a selected VH CDR3 amino acid sequence, and a lightchain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1region comprises or consists of an amino acid sequence that is at least80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acidsequence, the CDR2 region comprises or consists of an amino acidsequence that is at least 80%, 85%, 90%, or 95% identical to a selectedVL CDR2 amino acid sequence, and the CDR3 region comprises or consistsof an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected VHCDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3amino acid sequences are shown in FIG. 16(Kabat CDR) and FIG. 17(Chothia CDR).

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 2 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 3 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 8 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 9 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 13 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 14 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 17 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 3 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 18 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 19 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 9 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a heavy chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 20 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 21 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 14 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 5 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 6 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 11 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 12 with zero, one or two amino acid insertions, deletions, orsubstitutions.

In some embodiments, the antibody or an antigen-binding fragmentdescribed herein can contain a light chain variable domain containingone, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or twoamino acid insertions, deletions, or substitutions; SEQ ID NO: 11 withzero, one or two amino acid insertions, deletions, or substitutions; SEQID NO: 15 with zero, one or two amino acid insertions, deletions, orsubstitutions.

The insertions, deletions, and substitutions can be within the CDRsequence, or at one or both terminal ends of the CDR sequence.

The disclosure also provides antibodies or antigen-binding fragmentsthereof that bind to PD-1. The antibodies or antigen-binding fragmentsthereof contain a heavy chain variable region (VH) comprising orconsisting of an amino acid sequence that is at least 80%, 85%, 90%, or95% identical to a selected VH sequence, and a light chain variableregion (VL) comprising or consisting of an amino acid sequence that isat least 80%, 85%, 90%, or 95% identical to a selected VL sequence. Insome embodiments, the selected VH sequence is SEQ ID NOs: 26, 27, 28, or39, and the selected VL sequence is SEQ ID NOs: 29, 30, 31, or 40. Insome embodiments, the selected VH sequence is SEQ ID NOs: 32, 33, 34,35, or 41 and the selected VL sequence is SEQ ID NOs: 36, 37, 38, or 42.In some embodiments, the selected VH sequence is SEQ ID NO: 43, and theselected VL sequence is SEQ ID NO: 44.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 80% of the length of the reference sequence, and in someembodiments is at least 90%, 95%, or 100%. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. For purposes of the present disclosure, the comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The disclosure also provides nucleic acid comprising a polynucleotideencoding a polypeptide comprising an immunoglobulin heavy chain or animmunoglobulin light chain. The immunoglobulin heavy chain orimmunoglobulin light chain comprises CDRs as shown in FIG. 16 or FIG.17, or have sequences as shown in FIG. 19 or FIG. 20. When thepolypeptides are paired with corresponding polypeptide (e.g., acorresponding heavy chain variable region or a corresponding light chainvariable region), the paired polypeptides bind to PD-1 (e.g., humanPD-1).

The anti-PD-1 antibodies and antigen-binding fragments can also beantibody variants (including derivatives and conjugates) of antibodiesor antibody fragments and multi-specific (e.g., bi-specific) antibodiesor antibody fragments. Additional antibodies provided herein arepolyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific),human antibodies, chimeric antibodies (e.g., human-mouse chimera),single-chain antibodies, intracellularly-made antibodies (i.e.,intrabodies), and antigen-binding fragments thereof. The antibodies orantigen-binding fragments thereof can be of any type (e.g., IgG, IgE,IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, andIgA2), or subclass. In some embodiments, the antibody or antigen-bindingfragment thereof is an IgG antibody or antigen-binding fragment thereof.

Fragments of antibodies are suitable for use in the methods provided solong as they retain the desired affinity and specificity of thefull-length antibody. Thus, a fragment of an antibody that binds to PD-1will retain an ability to bind to PD-1. An Fv fragment is an antibodyfragment which contains a complete antigen recognition and binding site.This region consists of a dimer of one heavy and one light chainvariable domain in tight association, which can be covalent in nature,for example in scFv. It is in this configuration that the three CDRs ofeach variable domain interact to define an antigen binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs or a subsetthereof confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeCDRs specific for an antigen) can have the ability to recognize and bindantigen, although usually at a lower affinity than the entire bindingsite.

Single-chain Fv or (scFv) antibody fragments comprise the VH and VLdomains (or regions) of antibody, wherein these domains are present in asingle polypeptide chain. Generally, the scFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains, whichenables the scFv to form the desired structure for antigen binding.

The Fab fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′)₂ antibody fragments comprise a pair of Fab fragmentswhich are generally covalently linked near their carboxy termini byhinge cysteines between them. Other chemical couplings of antibodyfragments are also known in the art.

Diabodies are small antibody fragments with two antigen-binding sites,which fragments comprise a VH connected to a VL in the same polypeptidechain (VH and VL). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites.

Linear antibodies comprise a pair of tandem Fd segments (VH—CH1-VH—CH1)which, together with complementary light chain polypeptides, form a pairof antigen binding regions. Linear antibodies can be bispecific ormonospecific.

Antibodies and antibody fragments of the present disclosure can bemodified in the Fc region to provide desired effector functions or serumhalf-life.

Multimerization of antibodies may be accomplished through naturalaggregation of antibodies or through chemical or recombinant linkingtechniques known in the art. For example, some percentage of purifiedantibody preparations (e.g., purified IgG₁ molecules) spontaneously formprotein aggregates containing antibody homodimers and other higher-orderantibody multimers.

Alternatively, antibody homodimers may be formed through chemicallinkage techniques known in the art. For example, heterobifunctionalcrosslinking agents including, but not limited to SMCC (succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidylS-acetylthio-acetate) can be used to form antibody multimers. Anexemplary protocol for the formation of antibody homodimers is describedin Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997).Antibody homodimers can be converted to Fab′₂ homodimers throughdigestion with pepsin. Another way to form antibody homodimers isthrough the use of the autophilic T15 peptide described in Zhao et al.(J. Immunol. 25:396-404, 2002).

In some embodiments, the multi-specific antibody is a bi-specificantibody. Bi-specific antibodies can be made by engineering theinterface between a pair of antibody molecules to maximize thepercentage of heterodimers that are recovered from recombinant cellculture. For example, the interface can contain at least a part of theCH3 domain of an antibody constant domain. In this method, one or moresmall amino acid side chains from the interface of the first antibodymolecule are replaced with larger side chains (e.g., tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g., alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers. This method is described, e.g., in WO 96/27011, which isincorporated by reference in its entirety.

Bi-specific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin and the other to biotin. Heteroconjugateantibodies can also be made using any convenient cross-linking methods.Suitable cross-linking agents and cross-linking techniques are wellknown in the art and are disclosed in U.S. Pat. No. 4,676,980, which isincorporated herein by reference in its entirety.

Methods for generating bi-specific antibodies from antibody fragmentsare also known in the art. For example, bi-specific antibodies can beprepared using chemical linkage. Brennan et al. (Science 229:81, 1985)describes a procedure where intact antibodies are proteolyticallycleaved to generate F(ab′)2 fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′ TNB derivatives isthen reconverted to the Fab′ thiol by reduction with mercaptoethylamine,and is mixed with an equimolar amount of another Fab′ TNB derivative toform the bi-specific antibody.

Any of the antibodies or antigen-binding fragments described herein maybe conjugated to a stabilizing molecule (e.g., a molecule that increasesthe half-life of the antibody or antigen-binding fragment thereof in asubject or in solution). Non-limiting examples of stabilizing moleculesinclude: a polymer (e.g., a polyethylene glycol) or a protein (e.g.,serum albumin, such as human serum albumin). The conjugation of astabilizing molecule can increase the half-life or extend the biologicalactivity of an antibody or an antigen-binding fragment in vitro (e.g.,in tissue culture or when stored as a pharmaceutical composition) or invivo (e.g., in a human).

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein can be conjugated to a therapeutic agent. Theantibody-drug conjugate comprising the antibody or antigen-bindingfragment thereof can covalently or non-covalently bind to a therapeuticagent. In some embodiments, the therapeutic agent is a cytotoxic orcytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoidssuch as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

Antibody Characteristics

The antibodies or antigen-binding fragments thereof described herein canblock the binding between PD-1 and PD-L1 and/or the binding between PD-1and PD-L2. In some embodiments, by binding to PD-1, the antibody caninhibitPD-1 signaling pathway and upregulates the immune response. Thus,in some embodiments, the antibodies or antigen-binding fragments thereofas described herein are PD-1 antagonist. In some embodiments, theantibodies or antigen-binding fragments thereof are PD-1 agonist.

In some embodiments, the antibodies or antigen-binding fragments thereofas described herein can increase immune response, activity or number ofT cells (e.g., CD8+ and/or CD4+ cells) by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or20 folds. In some embodiments, the antibodies or antigen-bindingfragments thereof as described herein can decrease the activity ornumber of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.

In some implementations, the antibody (or antigen-binding fragmentsthereof) specifically binds to PD-1 (e.g., human PD-1, monkey PD-1,mouse PD-1, and/or chimeric PD-1) with a dissociation rate (koff) ofless than 0.1 s⁻¹, less than 0.01 s⁻¹, less than 0.001 s⁻¹, less than0.0001 s⁻¹, or less than 0.00001 s⁻¹. In some embodiments, thedissociation rate (koff) is greater than 0.01 s⁻¹, greater than 0.001s⁻¹, greater than 0.0001 s⁻¹, greater than 0.00001 s⁻¹, or greater than0.000001 s⁻¹.

In some embodiments, kinetic association rates (kon) is greater than1×10²/Ms, greater than 1×10³/Ms, greater than 1×10⁴/Ms, greater than1×10⁵/Ms, or greater than 1×10⁶/Ms. In some embodiments, kineticassociation rates (kon) is less than 1×10⁵/Ms, less than 1×10⁶/Ms, orless than 1×10⁷/Ms.

Affinities can be deduced from the quotient of the kinetic rateconstants (KD=koff/kon). In some embodiments, KD is less than 1×10⁻⁶M,less than 1×10⁻⁷M, less than 1×10⁻⁸M, less than 1×10⁻⁹M, or less than1×10⁻¹⁰ M. In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM,15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.In some embodiments, KD is greater than 1×10⁻⁷M, greater than 1×10⁻⁸M,greater than 1×10⁻⁹M, greater than 1×10⁻¹⁰ M, greater than 1×10⁻¹¹M, orgreater than 1×10⁻¹²M. In some embodiments, the antibody binds to humanPD-1 with KD less than or equal to about 3 nM.

General techniques for measuring the affinity of an antibody for anantigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR).In some embodiments, the antibody binds to human PD-1 (SEQ ID NO: 22),monkey PD-1 (e.g., rhesus macaque PD-1, SEQ ID NO: 24), chimeric PD-1(SEQ ID NO: 25), and/or mouse PD-1 (SEQ ID NO: 23). In some embodiments,the antibody does not bind to human PD-1 (SEQ ID NO: 22), monkey PD-1(e.g., rhesus macaque PD-1, SEQ ID NO: 24;

-   -   cynomolgus PD-1), chimeric PD-1 (SEQ ID NO: 25), and/or mouse        PD-1 (SEQ ID NO: 23).

In some embodiments, thermal stabilities are determined. The antibodiesor antigen binding fragments as described herein can have a Tm greaterthan 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,or 95° C.

As IgG can be described as a multi-domain protein, the melting curvesometimes shows two transitions, with a first denaturation temperature,Tm D1, and a second denaturation temperature Tm D2. The presence ofthese two peaks often indicate the denaturation of the Fcdomains (Tm D1)and Fabdomains (Tm D2), respectively. When there are two peaks, Tmusually refers to Tm D2. Thus, in some embodiments, the antibodies orantigen binding fragments as described herein has a Tm D1 greater than60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or95° C. In some embodiments, the antibodies or antigen binding fragmentsas described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

In some embodiments, Tm, Tm D1, Tm D2 are less than 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

In some embodiments, the antibody has a tumor growth inhibitionpercentage (TGI %) that is greater than 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, or 200%. In some embodiments, the antibody has a tumor growthinhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%,120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI % canbe determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after thetreatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 monthsafter the treatment starts. As used herein, the tumor growth inhibitionpercentage (TGI %) is calculated using the following formula:

TGI(%)=[1−(Ti−T0)/(Vi−V0)]×100

Ti is the average tumor volume in the treatment group on day i. T0 isthe average tumor volume in the treatment group on day zero. Vi is theaverage tumor volume in the control group on day i. V0 is the averagetumor volume in the control group on day zero.

In some embodiments, the antibodies or antigen-binding fragments thereofas described herein are PD-1 antagonist. In some embodiments, theantibodies or antigen binding fragments decrease PD-1 signaltransduction in a target cell that expresses PD-1.

In some embodiments, the antibodies or antigen binding fragments enhanceCD4+ effector T cell function, for example, by increasing CD4+ effectorT cell proliferation and/or increasing gamma interferon production bythe CD4+ effector T cell (e.g., as compared to proliferation and/orcytokine production prior to treatment with the antibodies or antigenbinding fragments). In some embodiments, the cytokine is gammainterferon. In some embodiments, the antibodies or antigen bindingfragments increase number of intratumoral (infiltrating) CD4+ effector Tcells (e.g., total number of CD4+ effector T cells, or e.g., percentageof CD4+ cells in CD45+ cells), e.g., as compared to number ofintratumoral (infiltrating) CD4+ T cells prior to treatment withantibodies or antigen binding fragments. In some embodiments, theantibodies or antigen binding fragments increase number of intratumoral(infiltrating) CD4+ effector T cells that express gamma interferon(e.g., total gamma interferon expressing CD4+ cells, or e.g., percentageof gamma interferon expressing CD4+ cells in total CD4+ cells), e.g., ascompared to number of intratumoral (infiltrating) CD4+ T cells thatexpress gamma interferon prior to treatment.

In some embodiments, the antibodies or antigen binding fragmentsincrease number of intratumoral (infiltrating) CD8+ effector T cells(e.g., total number of CD8+ effector T cells, or e.g., percentage ofCD8+ in CD45+ cells), e.g., as compared to number of intratumoral(infiltrating) CD8+T effector cells prior to treatment. In someembodiments, the antibodies or antigen binding fragments increase numberof intratumoral (infiltrating) CD8+ effector T cells that express gammainterferon (e.g., percentage of CD8+ cells that express gamma interferonin total CD8+ cells), e.g., compared to number of intratumoral(infiltrating) CD8+ T cells that express gamma interferon prior totreatment with anti-human PD-1 antibody.

In some embodiments, the antibodies or antigen binding fragments enhancememory T cell function, for example by increasing memory T cellproliferation and/or increasing cytokine (e.g., gamma interferon)production by the memory cell.

In some embodiments, the antibodies or antigen binding fragments inhibitTreg function, for example, by decreasing Treg suppression of effector Tcell function (e.g., effector T cell proliferation and/or effector Tcell cytokine secretion). In some embodiments, the effector T cell is aCD4+ effector T cell. In some embodiments, the antibodies or antigenbinding fragments reduce the number of intratumoral (infiltrating) Treg(e.g., total number of Treg or e.g., percentage of Fox3p+ cells in CD4+cells).

In some embodiments, the antibodies or antigen binding fragments aredepleting anti-hPD-1 antibody (e.g., depletes cells that express humanPD-1). In some embodiments, the antibodies or antigen binding fragmentsdeplete cells that express human PD-1 in vitro. In some embodiments, thehuman PD-1 expressing cells are CD4+ effector T cells, or Treg cells. Insome embodiments, depleting is by ADCC and/or phagocytosis.

In some embodiments, the antibodies or antigen binding fragments have afunctional Fc region. In some embodiments, effector function of afunctional Fc region is antibody-dependent cell-mediated cytotoxicity(ADCC). In some embodiments, effector function of a functional Fc regionis phagocytosis. In some embodiments, effector function of a functionalFc region is ADCC and phagocytosis. In some embodiments, the Fc regionis human IgG1, human IgG2, human IgG3, or human IgG4.

In some embodiments, the antibodies or antigen binding fragments do notinduce apoptosis in PD-1-expressing cells (e.g., Treg).

In some embodiments, the antibodies or antigen binding fragments do nothave a functional Fc region. For example, the antibodies or antigenbinding fragments are Fab, Fab′, F(ab′)2, and Fv fragments.

Methods of Making Anti-PD-1 Antibodies

An isolated fragment of human PD-1 can be used as an immunogen togenerate antibodies using standard techniques for polyclonal andmonoclonal antibody preparation. Polyclonal antibodies can be raised inanimals by multiple injections (e.g., subcutaneous or intraperitonealinjections) of an antigenic peptide or protein. In some embodiments, theantigenic peptide or protein is injected with at least one adjuvant. Insome embodiments, the antigenic peptide or protein can be conjugated toan agent that is immunogenic in the species to be immunized. Animals canbe injected with the antigenic peptide or protein more than one time(e.g., twice, three times, or four times).

The full-length polypeptide or protein can be used or, alternatively,antigenic peptide fragments thereof can be used as immunogens. Theantigenic peptide of a protein comprises at least 8 (e.g., at least 10,15, 20, or 30) amino acid residues of the amino acid sequence of PD-1and encompasses an epitope of the protein such that an antibody raisedagainst the peptide forms a specific immune complex with the protein. Asdescribed above, the full length sequence of human PD-1 is known in theart (SEQ ID NO: 22).

An immunogen typically is used to prepare antibodies by immunizing asuitable subject (e.g., human or transgenic animal expressing at leastone human immunoglobulin locus). An appropriate immunogenic preparationcan contain, for example, a recombinantly-expressed or achemically-synthesized polypeptide (e.g., a fragment of human PD-1). Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or a similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a PD-1 polypeptide, or an antigenic peptidethereof (e.g., part of PD-1) as an immunogen. The antibody titer in theimmunized subject can be monitored over time by standard techniques,such as with an enzyme-linked immunosorbent assay (ELISA) using theimmobilized PD-1 polypeptide or peptide. If desired, the antibodymolecules can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein A of proteinG chromatography to obtain the IgG fraction. At an appropriate timeafter immunization, e.g., when the specific antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler et al. (Nature256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al.,Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985), or trioma techniques. The technology for producing hybridomas iswell known (see, generally, Current Protocols in Immunology, 1994,Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.).Hybridoma cells producing a monoclonal antibody are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide or epitope of interest, e.g., using a standard ELISAassay.

Variants of the antibodies or antigen-binding fragments described hereincan be prepared by introducing appropriate nucleotide changes into theDNA encoding a human, humanized, or chimeric antibody, orantigen-binding fragment thereof described herein, or by peptidesynthesis. Such variants include, for example, deletions, insertions, orsubstitutions of residues within the amino acids sequences that make-upthe antigen-binding site of the antibody or an antigen-binding domain.In a population of such variants, some antibodies or antigen-bindingfragments will have increased affinity for the target protein, e.g.,PD-1. Any combination of deletions, insertions, and/or combinations canbe made to arrive at an antibody or antigen-binding fragment thereofthat has increased binding affinity for the target. The amino acidchanges introduced into the antibody or antigen-binding fragment canalso alter or introduce new post-translational modifications into theantibody or antigen-binding fragment, such as changing (e.g., increasingor decreasing) the number of glycosylation sites, changing the type ofglycosylation site (e.g., changing the amino acid sequence such that adifferent sugar is attached by enzymes present in a cell), orintroducing new glycosylation sites.

Antibodies disclosed herein can be derived from any species of animal,including mammals. Non-limiting examples of native antibodies includeantibodies derived from humans, primates, e.g., monkeys and apes, cows,pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats,and rodents (e.g., rats, mice, hamsters and rabbits), includingtransgenic rodents genetically engineered to produce human antibodies.

Human and humanized antibodies include antibodies having variable andconstant regions derived from (or having the same amino acid sequence asthose derived from) human germline immunoglobulin sequences. Humanantibodies may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs.

A humanized antibody, typically has a human framework (FR) grafted withnon-human CDRs. Thus, a humanized antibody has one or more amino acidsequence introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed by e.g., substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody. These methods are described in e.g., Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988); each of which isincorporated by reference herein in its entirety. Accordingly,“humanized” antibodies are chimeric antibodies wherein substantiallyless than an intact human V domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically mouse antibodies in which some CDR residues andsome FR residues are substituted by residues from analogous sites inhuman antibodies.

The choice of human VH and VL domains to be used in making the humanizedantibodies is very important for reducing immunogenicity. According tothe so-called “best-fit” method, the sequence of the V domain of a mouseantibody is screened against the entire library of known human-domainsequences. The human sequence which is closest to that of the mouse isthen accepted as the human FR for the humanized antibody (Sims et al.,J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)).

It is further important that antibodies be humanized with retention ofhigh specificity and affinity for the antigen and other favorablebiological properties. To achieve this goal, humanized antibodies can beprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved.

Ordinarily, amino acid sequence variants of the human, humanized, orchimeric anti-PD-1 antibody will contain an amino acid sequence havingat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identitywith a sequence present in the light or heavy chain of the originalantibody.

Identity or homology with respect to an original sequence is usually thepercentage of amino acid residues present within the candidate sequencethat are identical with a sequence present within the human, humanized,or chimeric anti-PD-1 antibody or fragment, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity.

Additional modifications to the anti-PD-1 antibodies or antigen-bindingfragments can be made. For example, a cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have any increased half-life in vitro and/or in vivo. Homodimericantibodies with increased half-life in vitro and/or in vivo can also beprepared using heterobifunctional cross-linkers as described, forexample, in Wolff et al. (Cancer Res. 53:2560-2565, 1993).Alternatively, an antibody can be engineered which has dual Fc regions(see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230,1989).

In some embodiments, a covalent modification can be made to theanti-PD-1 antibody or antigen-binding fragment thereof. These covalentmodifications can be made by chemical or enzymatic synthesis, or byenzymatic or chemical cleavage. Other types of covalent modifications ofthe antibody or antibody fragment are introduced into the molecule byreacting targeted amino acid residues of the antibody or fragment withan organic derivatization agent that is capable of reacting withselected side chains or the N- or C-terminal residues.

In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e.g. complex, hybridand high mannose structures) as measured by MALDI-TOF mass spectrometry,as described in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues; or position 314 in Kabat numbering);however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants mayhave improved ADCC function. In some embodiments, to reduce glycanheterogeneity, the Fc region of the antibody can be further engineeredto replace the Asparagine at position 297 with Alanine (N297A).

In some embodiments, to facilitate production efficiency by avoidingFab-arm exchange, the Fc region of the antibodies was further engineeredto replace the serine at position 228 (EU numbering) of IgG4 withproline (S228P). A detailed description regarding 5228 mutation isdescribed, e.g., in Silva et al. “The S228P mutation prevents in vivoand in vitro IgG4 Fab-arm exchange as demonstrated using a combinationof novel quantitative immunoassays and physiological matrixpreparation.” Journal of Biological Chemistry 290.9 (2015): 5462-5469,which is incorporated by reference in its entirety.

Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., anexpression vectors) that include an isolated polynucleotide disclosedherein (e.g., a polynucleotide that encodes a polypeptide disclosedherein), host cells into which are introduced the recombinant vectors(i.e., such that the host cells contain the polynucleotide and/or avector comprising the polynucleotide), and the production of recombinantantibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one ormore polynucleotide(s) of interest to a host cell when the vector isintroduced to the host cell. An “expression vector” is capable ofdelivering and expressing the one or more polynucleotide(s) of interestas an encoded polypeptide in a host cell into which the expressionvector has been introduced. Thus, in an expression vector, thepolynucleotide of interest is positioned for expression in the vector bybeing operably linked with regulatory elements such as a promoter,enhancer, and/or a poly-A tail, either within the vector or in thegenome of the host cell at or near or flanking the integration site ofthe polynucleotide of interest such that the polynucleotide of interestwill be translated in the host cell introduced with the expressionvector.

A vector can be introduced into the host cell by methods known in theart, e.g., electroporation, chemical transfection (e.g., DEAE-dextran),transformation, transfection, and infection and/or transduction (e.g.,with recombinant virus). Thus, non-limiting examples of vectors includeviral vectors (which can be used to generate recombinant virus), nakedDNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expressionvectors associated with cationic condensing agents.

In some implementations, a polynucleotide disclosed herein (e.g., apolynucleotide that encodes a polypeptide disclosed herein) isintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus, or may use areplication defective virus. In the latter case, viral propagationgenerally will occur only in complementing virus packaging cells.Suitable systems are disclosed, for example, in Fisher-Hoch et al.,1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann.N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S.Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat.No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science,252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219;Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502;Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993,Cir. Res., 73:1202-1207. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692.The uptake of naked DNA may be increased by coating the DNA ontobiodegradable beads that are efficiently transported into the cells.

For expression, the DNA insert comprising an antibody-encoding orpolypeptide-encoding polynucleotide disclosed herein can be operativelylinked to an appropriate promoter (e.g., a heterologous promoter), suchas the phage lambda PL promoter, the E. coli lac, trp and tac promoters,the SV40 early and late promoters and promoters of retroviral LTRs, toname a few. Other suitable promoters are known to the skilled artisan.The expression constructs can further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs may include a translationinitiating at the beginning and a termination codon (UAA, UGA, or UAG)appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors can include at least one selectablemarker. Such markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture and tetracycline or ampicillinresistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces, andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells.Appropriate culture mediums and conditions for the host cells describedherein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Non-limiting bacterial promoters suitable for use include the E. colilacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, thelambda PR and PL promoters and the trp promoter. Suitable eukaryoticpromoters include the CMV immediate early promoter, the HSV thymidinekinase promoter, the early and late SV40 promoters, the promoters ofretroviral LTRs, such as those of the Rous sarcoma virus (RSV), andmetallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997).

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986), which is incorporated herein by reference in itsentirety.

Transcription of DNA encoding an antibody of the present disclosure byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act to increase transcriptional activity of apromoter in a given host cell-type. Examples of enhancers include theSV40 enhancer, which is located on the late side of the replicationorigin at base pairs 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide (e.g., antibody) can be expressed in a modified form,such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag,and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties can be added to the polypeptide tofacilitate purification. Such regions can be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

Methods of Treatment

The antibodies or antibody or antigen-binding fragments thereof of thepresent disclosure can be used for various therapeutic purposes. In oneaspect, the disclosure provides methods for treating a cancer in asubject, methods of reducing the rate of the increase of volume of atumor in a subject over time, methods of reducing the risk of developinga metastasis, or methods of reducing the risk of developing anadditional metastasis in a subject. In some embodiments, the treatmentcan halt, slow, retard, or inhibit progression of a cancer. In someembodiments, the treatment can result in the reduction of in the number,severity, and/or duration of one or more symptoms of the cancer in asubject.

In one aspect, the disclosure features methods that includeadministering a therapeutically effective amount of an antibody orantigen-binding fragment thereof disclosed herein to a subject in needthereof (e.g., a subject having, or identified or diagnosed as having, acancer), e.g., breast cancer (e.g., triple-negative breast cancer),carcinoid cancer, cervical cancer, endometrial cancer, glioma, head andneck cancer, liver cancer, lung cancer, small cell lung cancer,lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer,renal cancer, colorectal cancer, gastric cancer, testicular cancer,thyroid cancer, bladder cancer, urethral cancer, or hematologicmalignancy. In some embodiments, the cancer is unresectable melanoma ormetastatic melanoma, non-small cell lung carcinoma (NSCLC), small celllung cancer (SCLC), bladder cancer, or metastatic hormone-refractoryprostate cancer. In some embodiments, the subject has a solid tumor. Insome embodiments, the cancer is squamous cell carcinoma of the head andneck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer(TNBC), or colorectal carcinoma. In some embodiments, the subject hasHodgkin's lymphoma. In some embodiments, the subject has triple-negativebreast cancer (TNBC), gastric cancer, urothelial cancer, Merkel-cellcarcinoma, or head and neck cancer.

In some embodiments, the compositions and methods disclosed herein canbe used for treatment of patients at risk for a cancer. Patients withcancer can be identified with various methods known in the art.

As used herein, by an “effective amount” is meant an amount or dosagesufficient to effect beneficial or desired results including halting,slowing, retarding, or inhibiting progression of a disease, e.g., acancer. An effective amount will vary depending upon, e.g., an age and abody weight of a subject to which the antibody, antigen bindingfragment, antibody-encoding polynucleotide, vector comprising thepolynucleotide, and/or compositions thereof is to be administered, aseverity of symptoms and a route of administration, and thusadministration can be determined on an individual basis.

An effective amount can be administered in one or more administrations.By way of example, an effective amount of an antibody or an antigenbinding fragment is an amount sufficient to ameliorate, stop, stabilize,reverse, inhibit, slow and/or delay progression of a cancer in a patientor is an amount sufficient to ameliorate, stop, stabilize, reverse, slowand/or delay proliferation of a cell (e.g., a biopsied cell, any of thecancer cells described herein, or cell line (e.g., a cancer cell line))in vitro. As is understood in the art, an effective amount of anantibody or antigen binding fragment may vary, depending on, inter alia,patient history as well as other factors such as the type (and/ordosage) of antibody used.

Effective amounts and schedules for administering the antibodies,antibody-encoding polynucleotides, and/or compositions disclosed hereinmay be determined empirically, and making such determinations is withinthe skill in the art. Those skilled in the art will understand that thedosage that must be administered will vary depending on, for example,the mammal that will receive the antibodies, antibody-encodingpolynucleotides, and/or compositions disclosed herein, the route ofadministration, the particular type of antibodies, antibody-encodingpolynucleotides, antigen binding fragments, and/or compositionsdisclosed herein used and other drugs being administered to the mammal.Guidance in selecting appropriate doses for antibody or antigen bindingfragment can be found in the literature on therapeutic uses ofantibodies and antigen binding fragments, e.g., Handbook of MonoclonalAntibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J.,1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in HumanDiagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977,pp. 365-389.

A typical daily dosage of an effective amount of an antibody is 0.01mg/kg to 100 mg/kg. In some embodiments, the dosage can be less than 100mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments,the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage isabout 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the at least one antibody,antigen-binding fragment thereof, or pharmaceutical composition (e.g.,any of the antibodies, antigen-binding fragments, or pharmaceuticalcompositions described herein) and, optionally, at least one additionaltherapeutic agent can be administered to the subject at least once aweek (e.g., once a week, twice a week, three times a week, four times aweek, once a day, twice a day, or three times a day). In someembodiments, at least two different antibodies and/or antigen-bindingfragments are administered in the same composition (e.g., a liquidcomposition). In some embodiments, at least one antibody orantigen-binding fragment and at least one additional therapeutic agentare administered in the same composition (e.g., a liquid composition).In some embodiments, the at least one antibody or antigen-bindingfragment and the at least one additional therapeutic agent areadministered in two different compositions (e.g., a liquid compositioncontaining at least one antibody or antigen-binding fragment and a solidoral composition containing at least one additional therapeutic agent).In some embodiments, the at least one additional therapeutic agent isadministered as a pill, tablet, or capsule. In some embodiments, the atleast one additional therapeutic agent is administered in asustained-release oral formulation.

In some embodiments, the one or more additional therapeutic agents canbe administered to the subject prior to, or after administering the atleast one antibody, antigen-binding antibody fragment, or pharmaceuticalcomposition (e.g., any of the antibodies, antigen-binding antibodyfragments, or pharmaceutical compositions described herein). In someembodiments, the one or more additional therapeutic agents and the atleast one antibody, antigen-binding antibody fragment, or pharmaceuticalcomposition (e.g., any of the antibodies, antigen-binding antibodyfragments, or pharmaceutical compositions described herein) areadministered to the subject such that there is an overlap in thebioactive period of the one or more additional therapeutic agents andthe at least one antibody or antigen-binding fragment (e.g., any of theantibodies or antigen-binding fragments described herein) in thesubject.

In some embodiments, the subject can be administered the at least oneantibody, antigen-binding antibody fragment, or pharmaceuticalcomposition (e.g., any of the antibodies, antigen-binding antibodyfragments, or pharmaceutical compositions described herein) over anextended period of time (e.g., over a period of at least 1 week, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1year, 2 years, 3 years, 4 years, or 5 years). A skilled medicalprofessional may determine the length of the treatment period using anyof the methods described herein for diagnosing or following theeffectiveness of treatment (e.g., the observation of at least onesymptom of cancer). As described herein, a skilled medical professionalcan also change the identity and number (e.g., increase or decrease) ofantibodies or antigen-binding antibody fragments (and/or one or moreadditional therapeutic agents) administered to the subject and can alsoadjust (e.g., increase or decrease) the dosage or frequency ofadministration of at least one antibody or antigen-binding antibodyfragment (and/or one or more additional therapeutic agents) to thesubject based on an assessment of the effectiveness of the treatment(e.g., using any of the methods described herein and known in the art).

In some embodiments, one or more additional therapeutic agents can beadministered to the subject. The additional therapeutic agent cancomprise one or more inhibitors selected from the group consisting of aninhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, aninhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, aninhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of aphosphatidylinositol 3-kinase (PI3K), an inhibitor of an Akt, aninhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton'styrosine kinase (BTK), and an inhibitor of Isocitrate dehydrogenase 1(IDH1) and/or Isocitrate dehydrogenase 2 (IDH2). In some embodiments,the additional therapeutic agent is an inhibitor of indoleamine2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

In some embodiments, the additional therapeutic agent can comprise oneor more inhibitors selected from the group consisting of an inhibitor ofHER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2,an inhibitor of CHK1, an inhibitor of activated hedgehog signalingpathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise oneor more therapeutic agents selected from the group consisting ofTrabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib,Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin,Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib,Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, anHsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine,Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine,lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, andenzastaurin.

In some embodiments, the additional therapeutic agent can comprise oneor more therapeutic agents selected from the group consisting of anadjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1,an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17antagonist, an HVEM antagonist, an ICOS agonist, a treatment targetingCX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, atreatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and aSelectin agonist.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin,pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to thesubject.

In some embodiments, the additional therapeutic agent is an anti-OX40antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4antibody, or an anti-GITR antibody.

Methods of modifying and using the anti-PD-1 antibodies are described,e.g., in US20170247454, US 20170081409 A1, US 20170044259 A1, and US20160159905; each of which is incorporated herein by reference in itsentirety.

Pharmaceutical Compositions and Routes of Administration

Also provided herein are pharmaceutical compositions that contain atleast one (e.g., one, two, three, or four) of the antibodies orantigen-binding fragments described herein. Two or more (e.g., two,three, or four) of any of the antibodies or antigen-binding fragmentsdescribed herein can be present in a pharmaceutical composition in anycombination. The pharmaceutical compositions may be formulated in anymanner known in the art.

Pharmaceutical compositions are formulated to be compatible with theirintended route of administration (e.g., intravenous, intraarterial,intramuscular, intradermal, subcutaneous, or intraperitoneal). Thecompositions can include a sterile diluent (e.g., sterile water orsaline), a fixed oil, polyethylene glycol, glycerine, propylene glycolor other synthetic solvents, antibacterial or antifungal agents, such asbenzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like, antioxidants, such as ascorbic acid or sodiumbisulfate, chelating agents, such as ethylenediaminetetraacetic acid,buffers, such as acetates, citrates, or phosphates, and isotonic agents,such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol orsorbitol), or salts (e.g., sodium chloride), or any combination thereof.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers (see, e.g., U.S. Pat. No. 4,522,811). Preparations of thecompositions can be formulated and enclosed in ampules, disposablesyringes, or multiple dose vials. Where required (as in, for example,injectable formulations), proper fluidity can be maintained by, forexample, the use of a coating, such as lecithin, or a surfactant.Absorption of the antibody or antigen-binding fragment thereof can beprolonged by including an agent that delays absorption (e.g., aluminummonostearate and gelatin). Alternatively, controlled release can beachieved by implants and microencapsulated delivery systems, which caninclude biodegradable, biocompatible polymers (e.g., ethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

Compositions containing one or more of any of the antibodies orantigen-binding fragments described herein can be formulated forparenteral (e.g., intravenous, intraarterial, intramuscular,intradermal, subcutaneous, or intraperitoneal) administration in dosageunit form (i.e., physically discrete units containing a predeterminedquantity of active compound for ease of administration and uniformity ofdosage).

Toxicity and therapeutic efficacy of compositions can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals (e.g., monkeys). One can, for example, determine the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population): the therapeuticindex being the ratio of LD50:ED50. Agents that exhibit high therapeuticindices are preferred. Where an agent exhibits an undesirable sideeffect, care should be taken to minimize potential damage (i.e., reduceunwanted side effects). Toxicity and therapeutic efficacy can bedetermined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used informulating an appropriate dosage of any given agent for use in asubject (e.g., a human). A therapeutically effective amount of the oneor more (e.g., one, two, three, or four) antibodies or antigen-bindingfragments thereof (e.g., any of the antibodies or antibody fragmentsdescribed herein) will be an amount that treats the disease in a subject(e.g., kills cancer cells) in a subject (e.g., a human subjectidentified as having cancer), or a subject identified as being at riskof developing the disease (e.g., a subject who has previously developedcancer but now has been cured), decreases the severity, frequency,and/or duration of one or more symptoms of a disease in a subject (e.g.,a human). The effectiveness and dosing of any of the antibodies orantigen-binding fragments described herein can be determined by a healthcare professional or veterinary professional using methods known in theart, as well as by the observation of one or more symptoms of disease ina subject (e.g., a human). Certain factors may influence the dosage andtiming required to effectively treat a subject (e.g., the severity ofthe disease or disorder, previous treatments, the general health and/orage of the subject, and the presence of other diseases).

Exemplary doses include milligram or microgram amounts of any of theantibodies or antigen-binding fragments described herein per kilogram ofthe subject's weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; or about 1μg/kg to about 50 μg/kg). While these doses cover a broad range, one ofordinary skill in the art will understand that therapeutic agents,including antibodies and antigen-binding fragments thereof, vary intheir potency, and effective amounts can be determined by methods knownin the art. Typically, relatively low doses are administered at first,and the attending health care professional or veterinary professional(in the case of therapeutic application) or a researcher (when stillworking at the development stage) can subsequently and graduallyincrease the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, andthe half-life of the antibody or antibody fragment in vivo.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. The disclosurealso provides methods of manufacturing the antibodies or antigen bindingfragments thereof for various uses as described herein.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. Generating Mouse Anti-hPD-1 Antibodies

To generate mouse antibodies against human PD-1 (hPD-1; SEQ ID NO: 22),6-8 weeks old female BALB/c mice were immunized with human PD-1.Anti-hPD-1 antibodies were collected by the methods as described belowand shown in FIG. 1 and FIG. 2.

Immunization of Mice

6-8 weeks old female BALB/c mice were immunized with His-tagged humanPD-1 proteins at 20 μg/mouse at a concentration of 100 μg/ml. TheHis-tagged human PD-1 proteins were emulsified with adjuvant andinjected at four positions on the back of the mice. For the firstsubcutaneous (s.c.) injection, the diluted antigen was emulsified withComplete Freund's Adjuvant (CFA) in equal volume. In the followingsubcutaneous injections, the protein was emulsified with IncompleteFreund's Adjuvant (IFA) in equal volume. Three days after the thirdinjection or theboosterimmunization, blood (serum) was collected andanalyzed for antibody titer using ELISA.

In another experiment, 6-8 weeks old female BALB/c mice were immunizedby injecting the expression plasmid encoding human PD-1 into the mice.The plasmids encoding the antigen were injected into the tibialisanterior muscle (intramuscular injection; i.m. injection) of the mice byusing gene guns at the concentration of 1000 μg/ml at 60 μg per mouse.At least four injections were performed with at least 14 days betweentwo injections. Blood (serum) was collected seven days after the lastimmunization and the serum was tested for antibody titer by ELISA.

Procedures to enhance immunization were also performed at least fourteendays after the previous immunization (either by injecting the plasmid orby injecting the proteins). CHO cells that express PD-1 antigen on thesurface were intravenously injected into the mice through tail veins.Spleen was then collected four days after the injection.

Fusion of SP2/0 Cells and Spleen Cells

Spleen tissues were grinded. Spleen cells were first selected by CD3cMicrobeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0cells. The cells were then plated in 96-well plates withhypoxanthine-aminopterin-thymidine (HAT) medium.

Primary Screening of Hybridoma

Primary screening of the hybridoma supernatant in the 96-well plates wasperformed using Fluorescence-Activated Cell Sorting (FACS) pursuant tostandard procedures. Chinese hamster ovary (CHO) cells were added to96-well plates (2×10⁴ cells per well) before the screening. 50 μl ofsupernatant was used. The antibodies that were used in experiments were

-   -   (1) Fluorescein (FITC)-conjugated AffiniPure F(ab)₂ Fragment        Goat Anti-Mouse IgG, Fcγ Fragment Specific, and    -   (2) Alexa Fluor® 647-conjugated AffiniPure F(ab)2 Fragment Goat        Anti-Human IgG, Fcγ Fragment Specific.

Sub-Cloning

Sub-cloning was performed using ClonePix® 2. In short, the positivewells identified during the primary screening were transferred tosemisolid medium, and IgG positive clones were identified and tested.FITC anti-mouse IgG Fc antibody was used.

Ascites Fluid Antibodies

1×10⁶ positive hybridoma cells were injected intraperitoneally to B-NDG®mice (Beijing Biocytogen, Beijing, China). Monoclonal antibodies wereproduced by growing hybridoma cells within the peritoneal cavity of themouse. The hybridoma cells multiplied and produced ascites fluid in theabdomens of the mice. The fluid contained a high concentration ofantibody which can be harvested for later use.

Purification of Antibodies

Antibodies in ascites fluid were purified using GE AKTA™ proteinchromatography (GE Healthcare, Chicago, Ill., United States). 25-1A7(“1A7”), 18-3F1 (“3F1”), and 3-6G1 (“6G1”) were among the mouseantibodies produced by the methods described above.

The VH, VL and CDR regions of the antibodies were determined. The heavychain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acidsequences of 1A7 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQID NOs: 16, 17, 3, 4, 5, 6 (Chothia numbering).

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3amino acid sequences of 3F1 are shown in SEQ ID NOs: 7-12 (Kabatnumbering) or SEQ ID NOs: 18, 19, 9, 10, 11, 12 (Chothia numbering).

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3amino acid sequences of 6G1 are shown in SEQ ID NOs: 7, 13, 14, 10, 11,15 (Kabat numbering) or SEQ ID NOs: 20, 21, 14, 10, 11, 15 (Chothianumbering).

Example 2. Humanization of the Mice Antibodies

The starting point for humanization was the mouse antibodies (e.g., 1A7and 3F1). The amino acid sequences for the heavy chain variable regionand the light chain variable region of these mouse antibodies weredetermined.

Three humanized heavy chain variable region variants (SEQ ID NOs: 26-28)and three humanized light chain variable region variants (SEQ ID NOs:29-31) for 1A7 were constructed, containing different modifications orsubstitutions.

Four humanized heavy chain variable region variants (SEQ ID NOs: 32-35)and three humanized light chain variable region variants (SEQ ID NOs:36-38) for 3F1 were constructed, containing different modifications orsubstitutions.

These humanized heavy chain variable region variants can be combinedwith any of the light chain variable region variants based on the samemouse antibody. For example, 1A7-H1 (SEQ ID NO: 26) can be combined withany humanized light chain variable region variant based on the samemouse antibody 1A7 (e.g., 1A7-K3 (SEQ ID NO: 31)), and the antibody islabeled accordingly (e.g., 1A7-H1K3).

These humanized antibodies were then generated with the use ofBioLuminate® 1.0 (Schrodinger, Shanghai, China).

Example 3. In Vitro Testing of the Mouse Anti-hPD-1 Antibodies: Blockingthe Binding of Human PD-1 (hPD-1) and Human PD-L1 (hPD-L1)

Blocking assays were performed to determine whether the anti-hPD-1antibodies can block the binding between hPD-1 and its ligand hPD-L1.

The anti-hPD-1 antibodies were collected from mouse ascites fluid andpurified by chromatography. 25 μl CHO cells transiently transfected withhuman PD-1 were added to each well in a plate. The purified antibodieswere titrated to final concentrations of 50, 5, 0.5, 0.05, 0.005 μg/ml.The titrated antibodies were added to each well at 25 μl per well at 4°C. and incubated for 30 minutes.

50 μl of Bitoin-hPD-L1 was added to each well (with a finalconcentration of 2 μg/ml in each well). The cells with Bitoin-hPD-L1 andthe antibodies were incubated at 4° C. for 15 minutes.

After being washed with phosphate-buffered saline (PBS) twice, 50 μl ofFITC labeled anti-mouse IgG Fc antibody (anti-mIgG Fc-FITC) at 1:100dilution and PE-labeled Streptavidin (Streptavidin-PE) at 1:100 dilutionwere added into each well, and incubated for 30 minutes at 4° C.,followed by PBS wash. The signals for FITC and PE were determined byflow cytometry.

As shown in FIG. 3, when the concentration of the mouse anti-PD-1antibodies 25-1A7, 18-3F1, and 03-6G1 increased, the signal for cellsbinging to PD-L1 decreased (y axis), while the signal for cells bindingto the mouse ant-PD-1 antibodies increased, suggesting that the bindingbetween human PD-1 and PD-L1 was blocked by the anti-hPD-1 antibodies.

Example 4. Cross-Reactivity of Anti-hPD-1 Antibodies Against Monkey,Mouse, and Human-Mouse Chimeric PD-1

In each experiment, the CHO cells were transfected with mouse PD-1(mPD-1, SEQ ID NO: 23), monkey (rhesus macaque) PD-1 (rmPD-1, SEQ ID NO:24), or chimeric (mouse and human) PD-1 (chiPD-1, SEQ ID NO: 25).

25 μl CHO cells were added to each well. 25 μl purified anti-hPD-1antibodies (1 μg/ml) (1A7, 3F1, or 6G1) were added to each well and wereincubated at 4° C. for 30 minutes.

After being washed with PBS (1200 rmp, 5 min) twice, 50 μl of FITClabeled anti-mouse IgG Fc antibody (anti-mIgG Fc-FITC) was added intoeach well 1:500 dilution, followed by incubated at 4° C. for 30 minutes,and then PBS wash (1200 rmp, 5 min). The signals for FITC weredetermined by flow cytometry.

As shown in FIG. 4, 1A7, 3F1, and 6G1 did not cross react with mousePD-1, but had strong cross reactivity with rmPD-1 and some crossreactivity with chimeric PD-1. In FIG. 4, NC stands for negativecontrol.

Example 5. Binding Affinity of Anti-hPD-1 Antibodies

The binding affinity of the anti-hPD-1 antibodies were measured usingsurface plasmon resonance (SPR) using Biacore™ (Biacore™, INC,Piscataway N.J.) T200 biosensor equipped with pre-immobilized Protein Asensor chips.

Anti-hPD-1 antibody 1A7-mHvKv-IgG4-S228P (1 μg/mL) were injected intoBiacore™ T200 biosensor at 10 μL/min for 30 seconds to achieve to adesired protein density (about 67 response units (RU)). Histidine-taggedhuman PD-1 proteins (hPD-1-His) at the concentration of 200, 100, 50,25, 12.5, 6.25, 3.125, 1.5625 nM were then injected at 30 μL/min for 100seconds. Dissociation was monitored for 400 seconds. The chip wasregenerated after the last injection of each titration with Glycine (pH2.0, 30 μL/min for 12 seconds). The result for 1A7-mHvKv-IgG4-S228P isshown in FIG. 5.

Kinetic association rates (kon) and dissociation rates (koff) wereobtained simultaneously by fitting the data globally to a 1:1 Langmuirbinding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994.Methods Enzymology 6. 99-110) using Biacore™ T200 Evaluation Software3.0. Affinities were deduced from the quotient of the kinetic rateconstants (KD=koff/kon).

As a person of ordinary skill in the art would understand, the samemethod with appropriate adjustments for parameters (e.g., antibodyconcentration) was performed for each tested antibody. For example, thegraph showing the results of 3F1-mHvKv-IgG4-S228P is shown in FIG. 6.The results for the tested antibodies are summarized in Table 1 below.

TABLE 1 Association rate Dissociation rate Affinity Anti-hPD-1antibodies kon (1/Ms) koff (1/s) KD (M) 1A7-mHvKv-IgG4-S228P 4.07E+059.56E−04 2.35E−09 1A7-H1K1-IgG4-S228P 5.68E+04 2.72E−03 4.79E−081A7-H1K2-IgG4-S228P 8.26E+04 2.65E−03 3.21E−08 1A7-H1K3-IgG4-S228P1.18E+05 1.19E−03 1.01E−08 1A7-H2K1-IgG4-S228P 4.89E+04 2.80E−035.73E−08 1A7-H2K2-IgG4-S228P 4.88E+04 2.82E−03 5.78E−081A7-H2K3-IgG4-S228P 1.15E+05 1.36E−03 1.19E−08 1A7-H3K1-IgG4-S228P6.62E+04 2.64E−03 3.99E−08 1A7-H3K2-IgG4-S228P 4.81E+04 2.80E−035.82E−08 1A7-H3K3-IgG4-S228P 1.02E+05 1.32E−03 1.29E−083F1-mHvKv-IgG4-S228P 3.70E+03 1.28E−03 3.46E−07 3F1-H1K1-IgG4-S228P4.81E+03 1.81E−03 3.76E−07 3F1-H1K2-IgG4-S228P 5.27E+03 1.55E−032.94E−07 3F1-H1K3-IgG4-S228P 4.79E+03 1.32E−03 2.75E−073F1-H2K1-IgG4-S228P 4.55E+03 1.92E−03 4.22E−07 3F1-H2K2-IgG4-S228P4.81E+03 1.70E−03 3.54E−07 3F1-H2K3-IgG4-S228P 4.41E+03 1.45E−033.29E−07 3F1-H3K1-IgG4-S228P 4.53E+03 1.79E−03 3.95E−073F1-H3K2-IgG4-S228P 5.06E+03 1.50E−03 2.96E−07 3F1-H3K3-IgG4-S228P4.38E+03 1.39E−03 3.18E−07 3F1-H4K1-IgG4-S228P 4.42E+03 2.04E−034.60E−07 3F1-H4K2-IgG4-S228P 4.80E+03 1.77E−03 3.69E−073F1-H4K3-IgG4-S228P 4.06E+03 1.61E−03 3.97E−07

Among these tested antibodies, 1A7 and 3F1 are mouse anti-hPD-1antibodies as described in Example 1. Based on 1A7 and 3F1, the chimericanti-hPD-1 antibodies including 1A7-mHvKv-IgG4-S228P, and3F1-mHvKv-IgG4-S228P were generated. The chimeric antibodies have theheavy chain variable domain and the light chain variable domain from thecorresponding mouse anti-hPD-1 antibodies, with the constant domainsfrom human IgG4 antibody (including, e.g., the CL, CH1, CH2, and CH3domains).

The tested antibodies also include humanized antibodies, e.g.,1A7-H1K1-IgG4-S228P, 1A7-H1K2-IgG4-S228P, 1A7-H1K3-IgG4-S228P,1A7-H2K1-IgG4-S228P, 1A7-H2K2-IgG4-S228P, 1A7-H2K3-IgG4-S228P,1A7-H3K1-IgG4-S228P, 1A7-H3K2-IgG4-S228P, 1A7-H3K3-IgG4-S228P,3F1-H1K1-IgG4-S228P, 3F1-H1K2-IgG4-S228P, 3F1-H1K3-IgG4-S228P,3F1-H2K1-IgG4-S228P, 3F1-H2K2-IgG4-S228P, 3F1-H2K3-IgG4-S228P,3F1-H3K1-IgG4-S228P, 3F1-H3K2-IgG4-S228P, 3F1-H3K3-IgG4-S228P,3F1-H4K1-IgG4-S228P, 3F1-H4K2-IgG4-S228P, and 3F1-H4K3-IgG4-S228P. Thehumanized antibodies have human IgG4 antibody constant domains(including, e.g., the CL, CH1, CH2, and CH3 domains). The humanizedvariable domains of the heavy chain are numbered H1, H2, H3 etc.; andthe humanized variable domains of the light chain are numbered K1, K2,K3 etc. The sequences of the humanized variable domains are summarizedin FIG. 19. For example, 1A7-H1K1-IgG4-S228P is based on the mouseantibody 1A7 and has the humanized heavy chain variable domain H1 (SEQID NO: 26) and humanized light chain variable domain K1 (SEQ ID NO: 29).Similarly, 3F1-H1K1-IgG4-5228P is based on mouse antibody 3F1 and hashumanized heavy chain variable domain H1 (SEQ ID NO: 32) and humanized9H3 light chain variable domain K1 (SEQ ID NO: 36).

The name and the sequences of the chimeric anti-PD-1 antibodies and thehumanized anti-PD-1 antibodies are summarized in Table 2 below. Tofacilitate production efficiency by avoiding Fab-arm exchange, the Fcregion of the antibodies was further engineered to replace the serine atposition 228 (EU numbering) with proline (S228P).

TABLE 2 VH SEQ VL SEQ Type Antibody name ID NO: ID NO: Constant regionsChimeric 1A7-m HyKy-IgG4- 39 40 Human IgG4 with S228P mutation antibodyS228P based on 1A7 Humanized 1A7-H1K1-IgG4-S228P 26 29 Human IgG4 withS228P mutation antibodies 1A7-H1K2-IgG4-S228P 26 30 Human IgG4 withS228P mutation based on 1A7-H1K3-IgG4-S228P 26 31 Human IgG4 with S228Pmutation 1A7 1A7-H2K1-IgG4-S228P 27 29 Human IgG4 with S228P mutation1A7-H2K2-IgG4-S228P 27 30 Human IgG4 with S228P mutation1A7-H2K3-IgG4-S228P 27 31 Human IgG4 with S228P mutation1A7-H3K1-IgG4-S228P 28 29 Human IgG4 with S228P mutation1A7-H3K2-IgG4-S228P 28 30 Human IgG4 with S228P mutation1A7-H3K3-IgG4-S228P 28 31 Human IgG4 with S228P mutation Chimeric3F1-mHyKy-IgG4- 41 42 Human IgG4 with S228P mutation antibody S228Pbased on 3F1 Humanized 3F1-H1K1-IgG4-S228P 32 36 Human IgG4 with S228Pmutation antibodies 3F1-H1K2-IgG4-S228P 32 37 Human IgG4 with S228Pmutation based on 3F1-H1K3-IgG4-S228P 32 38 Human IgG4 with S228Pmutation 3F1 3F1-H2K1-IgG4-S228P 33 36 Human IgG4 with S228P mutation3F1-H2K2-IgG4-S228P 33 37 Human IgG4 with S228P mutation3F1-H2K3-IgG4-S228P 33 38 Human IgG4 with S228P mutation3F1-H3K1-IgG4-S228P 34 36 Human IgG4 with S228P mutation3F1-H3K2-IgG4-S228P 34 37 Human IgG4 with S228P mutation3F1-H3K3-IgG4-S228P 34 38 Human IgG4 with S228P mutation3F1-H4K1-IgG4-S228P 35 36 Human IgG4 with S228P mutation3F1-H4K2-IgG4-S228P 35 37 Human IgG4 with S228P mutation3F1-H4K3-IgG4-S228P 35 38 Human IgG4 with S228P mutation

Example 6. Thermal Stability of Anti-hPD-1 Antibodies

Thermofluor assay was performed using the Protein Thermal Shift™ Dye Kit(Thermo Fisher Scientific) and QuantStudio™ 5 Real Time PCR Systems(Thermo Fisher Scientific). This assay measured thermostability using afluorescent dye that binds to hydrophobic patches exposed as the proteinunfolds.

The experiments were performed according to the manufacturer's protocol.2 μL of antibody, 10.5 μL of water, 5 μL of Protein Thermal Shiftbuffer, and 2.5 μl of diluted Protein Thermal Shift Dye were mixed.Samples were heated to 25° C. at 1.6° C./second, and then heated to 99°C. at 0.05° C./second.

The table below summarizes the Tm for four humanized anti-hPD-1antibodies.

TABLE 3 Thermal Variable Type stability Antibody Domains (constantdomains) (Tm° C.) 1A7-H1K3-IgG4-S228P 1A7 H1K3 Human IgG4 with 75.94S228P mutation 1A7-H2K3-IgG4-S228P 1A7 H2K3 Human IgG4 with 76.09 S228Pmutation 3F1-H1K2-IgG4-S228P 3F1 H1K2 Human IgG4 with 82.82 S228Pmutation 3F1-H1K3-IgG4-S228P 3F1 H1K3 Human IgG4 with 82.37 S228Pmutation

Example 7. In Vivo Testing of Mouse and Chimeric Anti-hPD-1 Antibodies

In order to test the anti-hPD-1 antibodies in vivo and to predict theeffects of these antibodies in human, a humanized PD-1 mouse model wasgenerated. The humanized PD-1 mouse model was engineered to express achimeric PD-1 protein (SEQ ID NO: 25) wherein a part of theextracellular region of the mouse PD-1 protein was replaced with thecorresponding human PD-1 extracellular region. The amino acid residues31-141 of mouse PD-1 (SEQ ID NO: 23) were replaced by amino acidresidues 31-141 of human PD-1 (SEQ ID NO: 22). The humanized mouse model(B-hPD-1) provides a new tool for testing new therapeutic treatments ina clinical setting by significantly decreasing the difference betweenclinical outcome in human and in ordinary mice expressing mouse PD-1. Adetailed description regarding humanized PD-1 mouse model can be foundin PCT/CN2017/090320, which is incorporated herein by reference in itsentirety.

The anti-hPD-1 antibodies were tested for their effect on tumor growthin vivo in a model of colon carcinoma. MC-38 cancer tumor cells (colonadenocarcinoma cell) were injected subcutaneously in B-hPD-1 mice. Whenthe tumors in the mice reached a volume of 150±50 mm³, the mice wererandomly placed into different groups based on the volume of the tumor(five mice in each group).

The mice were then injected with physiological saline (PS) andanti-hPD-1 antibodies by intraperitoneal administration. The antibodywas given on the first day and the fourth day of each week for 3 weeks(6 injections in total).

The injected volume was calculated based on the weight of the mouse at 1mg/kg. The length of the long axis and the short axis of the tumor weremeasured and the volume of the tumor was calculated as 0.5×(longaxis)×(short axis). The weight of the mice was also measured before theinjection, when the mice were placed into different groups (before thefirst antibody injection), twice a week during the antibody injectionperiod, and before euthanization.

The tumor growth inhibition percentage (TGI %) was calculated using thefollowing formula: TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100. Ti is the averagetumor volume in the treatment group on day i. T0 is the average tumorvolume in the treatment group on day zero. Vi is the average tumorvolume in the control group on day i. V0 is the average tumor volume inthe control group on day zero.

T-test was performed for statistical analysis. A TGI % higher than 60%indicates significant suppression of tumor growth. P<0.05 is a thresholdto indicate significant difference.

In Vivo Results for Mouse Anti-hPD-1 Antibodies 25-1A7 and 18-3F1

In each of the four groups (G1, G2, G3, G4), B-hPD-1 mice were injectedwith each physiological saline (PS) as a control (G1), Keytruda (G2),the mouse anti-hPD-1 antibody 25-1A7 (G3), or the mouse anti-hPD-1antibody 18-3F1 (G4). The weight of the mice was monitored during theentire treatment period. The weight of mice in different groups allincreased (FIG. 7 and FIG. 8). No significant difference in weight wasobserved among the four groups. The results showed that 1A7 and 3F1 werewell tolerated and not toxic to the mice.

The tumor size in groups treated with Keytruda, 1A7, or 3F1 increased toa lesser extent compared to the control group (FIG. 9). Particularly,the tumor size in G3 is smaller than G2; and the tumor size in G4 issmaller than G2.

The TGI % at day 24 (24 days after grouping) was also calculated asshown in the table below.

TABLE 4 Average P value tumor size at (against Group Antibodies Day 24TGI % control) G1 PS 1078 — — (1 mg/kg; i.p. administration; twice aweek, 6 injections in total) G2 Keytruda(1 mg/kg) 842 25.3% 0.691 (1mg/kg; i.p. administration; twice a week, 6 injections in total) G325-1A7(1 mg/kg) 258 87.9% 0.153 (1 mg/kg; i.p. administration; twice aweek, 6 injections in total) G4 18-3F1(1 mg/kg) 646 46.3% 0.439 (1mg/kg; i.p. administration; twice a week, 6 injections in total)

In Vivo Results for Chimeric Anti-hPD-1 Antibodies

Chimeric anti-hPD-1 antibodies 03-6G1-mHvKv-IgG4-S228P (Group 2, G2),18-3F1-mHvKv-IgG4-S228P (Group 3, G3), 25-1A7-mHvKv-IgG4-S228P (Group 4,G4), and Keytruda (Group 5) were administered into B-hPD-1 mice(humanized PD-1 mice) by intraperitoneal administration. Physiologicalsaline was injected as a control (Group 1, G1). The injected amount ofthe antibodies was calculated based on the weight of the mouse at 1mg/kg. The antibodies were given on the first day and the fourth day ofeach week (6 injections in total).

The weight of the mice was monitored during the entire treatment period.The weight of mice in different groups all increased (FIG. 10, and FIG.11). No significant difference in weight was observed among thedifferent groups. The results showed that the anti-hPD-1 antibodies werewell tolerated and not toxic to the mice.

The tumor size showed significant difference in groups treated with thechimeric antibodies compared to the control group (FIG. 12).

The TGI % at day 20 (20 days after grouping) for each treatment groupwas calculated as shown in the table below.

TABLE 5 Average tumor size at Group Antibodies Day 20 TGI % P value G1PS 1427 — — G2 03-6G1-mHvKv-IgG4-S228P 564 66.0% 0.032 (1 mg/kg; i.p.administration; twice a week, 6 injections in total) G318-3F1-mHvKv-IgG4-S228P 700 55.6% 0.056 (1 mg/kg; i.p. administration;twice a week, 6 injections in total) G4 25-1A7-mHvKv-IgG4-S228P 35681.9% 0.011 (1 mg/kg; i.p. administration; twice a week, 6 injections intotal) G5 Keytruda 200 93.8% 0.005 (1 mg/kg; i.p. administration; twicea week, 6 injections in total)

The results showed that chimeric antibodies 03-6G1-mHvKv-IgG4-S228P and25-1A7-mHvKv-IgG4-S228P significantly inhibited tumor growth. Among thethree chimeric antibodies, 25-1A7-mHvKv-IgG4-S228P had the highest TGI%.

Example 8. In Vivo Testing of Humanized Anti-hPD-1 Antibodies

The humanized anti-hPD-1 antibodies were tested in PD-1 humanized mice(B-hPD-1) to demonstrate their effect on tumor growth in vivo.

MC-38 cancer tumor cells (colon adenocarcinoma cell) were injectedsubcutaneously in B-hPD-1 mice. When the tumors in the mice reached avolume of 150±50 mm³, the mice were randomly placed into differentgroups based on the volume of the tumor (five mice in each group).

The mice were then injected with physiological saline as a control (G1),humanized anti-PD-1 antibody 1A7-H1K3-IgG4-S228P (G2), humanizedanti-PD-1 antibody 1A7-H2K3-IgG4-S228P (G3), or Keytruda (G4). Theantibodies were given on the second day and the fifth day of each weekby intraperitoneal injection at 1 mg/kg for 3 weeks (6 injections intotal).

The weight of the mice was monitored during the entire treatment period.The weight of mice in different groups all increased (FIG. 13, and FIG.14). No significant difference in weight was observed among thedifferent groups. The results showed that the anti-hPD-1 antibodies werewell tolerated and not toxic to the mice.

The tumor size showed significant difference in groups treated with theanti-hPD-1 antibodies (FIG. 15). Particularly, the tumor size in G2 issmaller than G4; and the tumor size in G3 is smaller than G4.

The TGI % at Day 21 (21 days after grouping) for each treatment groupwas also calculated as shown in the table below.

TABLE 6 Average tumor size at Group Antibodies Day 18 TGI % P value G1PS 1986 — — G2 1A7-H1K3-IgG4-S228P 233 94.5% 0.001 (1 mg/kg) G31A7-H2K3-IgG4-S228P 150 99.0% 0.001 (1 mg/kg) G4 Keytruda 422 84.4%0.006 (1 mg/kg)

The results above show that all the anti-hPD-1 antibodies can inhibittumor growth. Among them, 1A7-H2K3-IgG4-S228P (1 mg/kg) had the highesttumor growth inhibition percentage (TGI %).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An antibody or antigen-binding fragment thereofthat binds to PD-1 (Programmed Cell Death Protein 1) comprising: a heavychain variable region (VH) comprising complementarity determiningregions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises anamino acid sequence that is at least 80% identical to a selected VH CDR1amino acid sequence, the VH CDR2 region comprises an amino acid sequencethat is at least 80% identical to a selected VH CDR2 amino acidsequence, and the VH CDR3 region comprises an amino acid sequence thatis at least 80% identical to a selected VH CDR3 amino acid sequence; anda light chain variable region (VL) comprising CDRs 1, 2, and 3, whereinthe VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 regioncomprises an amino acid sequence that is at least 80% identical to aselected VL CDR2 amino acid sequence, and the VL CDR3 region comprisesan amino acid sequence that is at least 80% identical to a selected VLCDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 aminoacid sequences and the selected VL CDRs, 1, 2, and 3 amino acidsequences are one of the following: (1) the selected VH CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively,and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 4, 5, 6, respectively; (2) the selected VH CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively,and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth inSEQ ID NOs: 10, 11, 12, respectively; (3) the selected VH CDRs 1, 2, 3amino acid sequences are set forth in SEQ ID NOs: 7, 13, 14,respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences areset forth in SEQ ID NOs: 10, 11, 15, respectively.
 2. The antibody orantigen-binding fragment thereof of claim 1, wherein the VH comprisesCDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1,2, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the aminoacid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. 3.The antibody or antigen-binding fragment thereof of claim 1, wherein theVH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQID NOs: 7, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12,respectively.
 4. The antibody or antigen-binding fragment thereof ofclaim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acidsequences set forth in SEQ ID NOs: 7, 13, and 14, respectively, and theVL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQID NOs: 10, 11, and 15, respectively.
 5. The antibody or antigen-bindingfragment thereof of any one of claims 1-4, wherein the antibody orantigen-binding fragment specifically binds to human PD-1.
 6. Theantibody or antigen-binding fragment thereof of any one of claims 1-5,wherein the antibody or antigen-binding fragment is a humanized antibodyor antigen-binding fragment thereof.
 7. The antibody or antigen-bindingfragment thereof of any one of claims 1-6, wherein the antibody orantigen-binding fragment is a single-chain variable fragment (scFV). 8.A nucleic acid comprising a polynucleotide encoding a polypeptidecomprising: (1) an immunoglobulin heavy chain or a fragment thereofcomprising a heavy chain variable region (VH) comprising complementaritydetermining regions (CDRs) 1, 2, and 3 comprising the amino acidsequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, andwherein the VH, when paired with a light chain variable region (VL)comprising the amino acid sequence set forth in SEQ ID NO: 29, 30, 31,or 40, binds to PD-1; (2) an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 comprising the aminoacid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, andwherein the VL, when paired with a VH comprising the amino acid sequenceset forth in SEQ ID NO: 26, 27, 28, or 39, binds to PD-1; (3) animmunoglobulin heavy chain or a fragment thereof comprising a heavychain variable region (VH) comprising CDRs 1, 2, and 3 comprising theamino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively,and wherein the VH, when paired with a light chain variable region (VL)comprising the amino acid sequence set forth in SEQ ID NO: 36, 37, 38 or42, binds to PD-1; (4) an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 comprising the aminoacid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively,and wherein the VL, when paired with a VH comprising the amino acidsequence set forth in SEQ ID NO: 32, 33, 34, 35 or 41, binds to PD-1;(5) an immunoglobulin heavy chain or a fragment thereof comprising aheavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprisingthe amino acid sequences set forth in SEQ ID NOs: 7, 13, and 14,respectively, and wherein the VH, when paired with a light chainvariable region (VL) comprising the amino acid sequence set forth in SEQID NO: 44 binds to PD-1; (6) an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 comprising the aminoacid sequences set forth in SEQ ID NOs: 10, 11, and 15, respectively,and wherein the VL, when paired with a VH comprising the amino acidsequence set forth in SEQ ID NO: 43 binds to PD-1.
 9. The nucleic acidof claim 8, wherein the nucleic acid comprises a polynucleotide encodinga polypeptide comprising an immunoglobulin heavy chain or a fragmentthereof comprising a VH comprising CDRs 1, 2, and 3 comprising the aminoacid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively. 10.The nucleic acid of claim 8, wherein the nucleic acid comprises apolynucleotide encoding a polypeptide comprising an immunoglobulin lightchain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and6, respectively.
 11. The nucleic acid of claim 8, wherein the nucleicacid comprises a polynucleotide encoding a polypeptide comprising animmunoglobulin heavy chain or a fragment thereof comprising a VHcomprising CDRs 1, 2, and 3 comprising the amino acid sequences setforth in SEQ ID NOs: 7, 8, and 9, respectively.
 12. The nucleic acid ofclaim 8, wherein the nucleic acid comprises a polynucleotide encoding apolypeptide comprising an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 comprising the aminoacid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.13. The nucleic acid of claim 8, wherein the nucleic acid comprises apolynucleotide encoding a polypeptide comprising an immunoglobulin heavychain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3comprising the amino acid sequences set forth in SEQ ID NOs: 7, 13, and14, respectively.
 14. The nucleic acid of claim 8, wherein the nucleicacid comprises a polynucleotide encoding a polypeptide comprising animmunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 comprising the amino acid sequences setforth in SEQ ID NOs: 10, 11, and 15, respectively.
 15. The nucleic acidof any one of claims 8-14, wherein the VH when paired with a VLspecifically binds to human PD-1, or the VL when paired with a VHspecifically binds to human PD-1.
 16. The nucleic acid of any one ofclaims 8-15, wherein the immunoglobulin heavy chain or the fragmentthereof is a humanized immunoglobulin heavy chain or a fragment thereof,and the immunoglobulin light chain or the fragment thereof is ahumanized immunoglobulin light chain or a fragment thereof.
 17. Thenucleic acid of any one of claims 8-16, wherein the nucleic acid encodesa single-chain variable fragment (scFv).
 18. The nucleic acid of any oneof claims 8-17, wherein the nucleic acid is cDNA.
 19. A vectorcomprising one or more of the nucleic acids of any one of claims 8-18.20. A vector comprising two of the nucleic acids of any one of claims8-18, wherein the vector encodes the VL region and the VH region thattogether bind to PD-1.
 21. A pair of vectors, wherein each vectorcomprises one of the nucleic acids of any one of claims 8-18, whereintogether the pair of vectors encodes the VL region and the VH regionthat together bind to PD-1.
 22. A cell comprising the vector of claim 19or 20, or the pair of vectors of claim
 21. 23. The cell of claim 22,wherein the cell is a CHO cell.
 24. A cell comprising one or more of thenucleic acids of any one of claims 8-18.
 25. A cell comprising two ofthe nucleic acids of any one of claims 8-18.
 26. The cell of claim 25,wherein the two nucleic acids together encode the VL region and the VHregion that together bind to PD-1.
 27. A method of producing an antibodyor an antigen-binding fragment thereof, the method comprising (a)culturing the cell of any one of claims 22-26 under conditionssufficient for the cell to produce the antibody or the antigen-bindingfragment; and (b) collecting the antibody or the antigen-bindingfragment produced by the cell.
 28. An antibody or antigen-bindingfragment thereof that binds to PD-1 comprising a heavy chain variableregion (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region(VL) comprising an amino acid sequence that is at least 90% identical toa selected VL sequence, wherein the selected VH sequence and theselected VL sequence are one of the following: (1) the selected VHsequence is SEQ ID NOs: 26, 27, 28, or 39, and the selected VL sequenceis SEQ ID NOs: 29, 30, 31, or 40; (2) the selected VH sequence is SEQ IDNOs: 32, 33, 34, 35 or 41, and the selected VL sequence is SEQ ID NOs:36, 37, 38 or 42; (3) the selected VH sequence is SEQ ID NO: 43, and theselected VL sequence is SEQ ID NO:
 44. 29. The antibody orantigen-binding fragment thereof of claim 28, wherein the VH comprisesthe sequence of SEQ ID NO: 26 and the VL comprises the sequence of SEQID NO:
 31. 30. The antibody or antigen-binding fragment thereof of claim28, wherein the VH comprises the sequence of SEQ ID NO: 27 and the VLcomprises the sequence of SEQ ID NO:
 31. 31. The antibody orantigen-binding fragment thereof of any one of claims 28-31, wherein theantibody or antigen-binding fragment specifically binds to human PD-1.32. The antibody or antigen-binding fragment thereof of any one ofclaims 28-32, wherein the antibody or antigen-binding fragment is ahumanized antibody or antigen-binding fragment thereof.
 33. The antibodyor antigen-binding fragment thereof of any one of claims 28-30, whereinthe antibody or antigen-binding fragment is a single-chain variablefragment (scFV).
 34. An antibody-drug conjugate comprising the antibodyor antigen-binding fragment thereof of any one of claims 1-7 and 28-33covalently bound to a therapeutic agent.
 35. The antibody drug conjugateof claim 34, wherein the therapeutic agent is a cytotoxic or cytostaticagent.
 36. A method of treating a subject having cancer, the methodcomprising administering a therapeutically effective amount of acomposition comprising the antibody or antigen-binding fragment thereofof any one of claims 1-7 and 28-33, or the antibody-drug conjugate ofclaim 34 or 35, to the subject.
 37. The method of claim 36, wherein thesubject has a solid tumor.
 38. The method of claim 36, wherein thecancer is unresectable melanoma or metastatic melanoma.
 39. The methodof claim 36, wherein the cancer is non-small cell lung cancer (NSCLC),squamous cell carcinoma of the head and neck (SCCHN), head and neckcancer, renal cell carcinoma (RCC), melanoma, bladder cancer, gastriccancer, urothelial cancer, Merkel-cell carcinoma, triple-negative breastcancer (TNBC), or colorectal carcinoma.
 40. A method of decreasing therate of tumor growth, the method comprising contacting a tumor cell withan effective amount of a composition comprising an antibody orantigen-binding fragment thereof of any one of claims 1-7 and 28-33, orthe antibody-drug conjugate of claims 34 or
 35. 41. A method of killinga tumor cell, the method comprising contacting a tumor cell with aneffective amount of a composition comprising the antibody orantigen-binding fragment thereof of any one of claims 1-7 and 28-33, orthe antibody-drug conjugate of claims 34 or
 35. 42. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragment thereofof any one of claims 1-7 and 28-33, and a pharmaceutically acceptablecarrier.
 43. A pharmaceutical composition comprising the antibody drugconjugate of claim 34 or 35, and a pharmaceutically acceptable carrier.